U.S. patent application number 15/175698 was filed with the patent office on 2016-09-29 for biomarker normalization.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Benjamin D. Sullivan.
Application Number | 20160282252 15/175698 |
Document ID | / |
Family ID | 46330245 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160282252 |
Kind Code |
A1 |
Sullivan; Benjamin D. |
September 29, 2016 |
BIOMARKER NORMALIZATION
Abstract
A fluid sample is measured with a tear film measuring system
that includes a processing device that receives a sample chip
comprising a sample region configured to contain an aliquot volume
of sample fluid, the processing device configured to perform
analyses of osmolarity and of one or more biomarkers within the
sample fluid, wherein the analysis of biomarkers includes
normalization of biomarker concentration values.
Inventors: |
Sullivan; Benjamin D.; (San
Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
46330245 |
Appl. No.: |
15/175698 |
Filed: |
June 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13842474 |
Mar 15, 2013 |
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15175698 |
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13299197 |
Nov 17, 2011 |
9217702 |
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13842474 |
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13031051 |
Feb 18, 2011 |
9217701 |
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13299197 |
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12104355 |
Apr 16, 2008 |
7905134 |
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13031051 |
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11358986 |
Feb 21, 2006 |
7574902 |
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12104355 |
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10400617 |
Mar 25, 2003 |
7017394 |
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11358986 |
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60912129 |
Apr 16, 2007 |
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60401432 |
Aug 6, 2002 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/487 20130101;
G01N 27/07 20130101; G01N 2800/24 20130101; G01N 21/6486 20130101;
B01L 2300/0654 20130101; B01L 3/502715 20130101; G01N 27/26
20130101; G01N 13/04 20130101; G01N 2800/162 20130101; G16B 40/00
20190201 |
International
Class: |
G01N 13/04 20060101
G01N013/04; B01L 3/00 20060101 B01L003/00; G01N 21/64 20060101
G01N021/64; G01N 33/487 20060101 G01N033/487 |
Claims
1-37. (canceled)
38. A fluid measuring system for measuring osmolarity of a sample
fluid and concentration of a biomarker within the sample fluid,
said system comprising: a) a sample chip comprising a sample fluid;
an optical energy source that illuminates the sample fluid; and an
optical detector that receives optical energy from the illuminated
sample fluid in response to imparting energy into the sample fluid,
and which processes (i) optical energy properties of the sample
fluid to prepare an osmolarity output signal which corresponds to
osmolarity of the sample fluid, and (ii) chemical properties of the
sample fluid to prepare a biomarker output signal which corresponds
to concentration of a biomarker within the sample fluid; and b) a
processing device that: receives the osmolarity output signal from
the sample fluid and processes the osmolarity output signal to
produce an osmolarity value for the sample fluid; and receives the
biomarker output signal from the sample fluid and processes the
biomarker output signal to produce a biomarker concentration value
for the sample fluid.
39. The fluid measuring system of claim 38, wherein the biomarker
is IgE, IgA, IgG, IgM, glucose, insulin, lactoferrin, a cytokine, a
hormone, a hormone metabolite, an infectious disease phenotype, a
nucleic acid, a protein, or a lipid fraction.
40. The fluid measuring system of claim 38, further comprising
normalization of biomarker concentration values.
41. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values corrects for
patient-specific tear homeostasis.
42. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values corrects for
clinician induced tear sampling variance in connection with
obtaining the sample fluid.
43. The fluid measuring system of claim 38, wherein the analyses of
osmolarity and of the biomarkers are performed simultaneously.
44. The fluid measuring system of claim 38, wherein the analyses of
osmolarity and of the biomarkers are performed serially.
45. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values is linear.
46. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values is ratiometric.
47. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values is exponential.
48. The fluid measuring system of claim 40, wherein the
normalization of biomarker concentration values is based on a
calibration curve.
49. A system as defined in claim 38, wherein the optical detector
includes a photodiode, wherein the optical detector includes a
photodiode.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
Non-Provisional application Ser. No. 11/358,986 entitled "Tear Film
Osmometer" filed on Feb. 21, 2006 (attorney docket no.
021935-000311 US), which is a continuation application of U.S.
Utility application Ser. No. 10/400,617 entitled "Tear Film
Osmometer" filed Mar. 25, 2003 (now U.S. Pat. No. 7,017,394), which
claims priority to U.S. Provisional Patent Application Ser. No.
60/401,432 entitled "Volume Independent Tear Film Osmometer" filed
Aug. 6, 2002. This application claims the benefit of priority under
35 U.S.C. 119 to U.S. Provisional Application Ser. No. 60/912,129
entitled "Biomarker Normalization" filed on Apr. 16, 2007 (attorney
docket no. 021935-001100US). Each of these applications is
incorporated herein in its entirety as if set forth in full.
BACKGROUND
[0002] 1. Field of the Invention
[0003] The field of the invention relates generally to osmolarity
measurements and more particularly to systems and methods for
calibrating tar film osmolarity measuring devices.
[0004] 2. Background Information
[0005] Tears fulfill an essential role in maintaining ocular
surface integrity, protecting against microbial challenge, and
preserving visual acuity. These functions, in turn, are critically
dependent upon the composition and stability of the tear film
structure, which includes an underlying mucin foundation, a middle
aqueous component, and an overlying lipid layer. Disruption,
deficiency, or absence of the tear film can severely impact the
eye. If unmanaged with artificial tear substitutes or tear film
conservation therapy, these disorders can lead to intractable
desiccation of the corneal epithelium, ulceration and perforation
of the cornea, an increased incidence of infections disease, and
ultimately pronounced visual impairment and blindness.
[0006] Keratoconjunctivitis sicca (KCS), or "dry eye", is a
condition in which one or more of the tear film structure
components listed above is present in insufficient volume or is
otherwise out of balance with the other components. It is known
that the (fluid tonicity or osmolarity of tears increases in
patients with KCS. KCS is associated with conditions that affect
the general health of the body, such as Sjogrcn's syndrome, aging,
and androgen deficiency. Therefore, osmolarity of a tear film can
be a sensitive and specific indicator for the diagnosis of KCS and
other conditions.
[0007] The osmolarity of a sample fluid (e.g., a tear) can be
determined by an ex vivo technique called "freezing point
depression," in which solutes or ions in a solvent (i.e. water),
cause a lowering of the fluid freezing point from what it would be
without the ions. In the freezing point depression analysis the
freezing point of the ionized sample fluid is found by detecting
the temperature at which a quantity of the sample (typically on the
order of about several milliliters) first begins to freeze in a
container (e.g., a tube). To measure the freezing point, a volume
of the sample fluid is collected into a container, such as a tube.
Next, a temperature probe is immersed in the sample fluid, and the
container is brought into contact with a freezing bath or Peltier
cooling device. The sample is continuously stirred so as to achieve
a supercooled liquid state below its freezing point. Upon
mechanical induction, the sample solidifies, rising to its freezing
point due to the thermodynamic heat of fusion. The deviation from
the sample freezing point from 0.degree. C. is proportional to the
solute level in the sample fluid. This type of measuring device is
sometimes referred to as an osmometer.
[0008] Presently, freezing point depression measurements are made
ex vivo by removing tear samples from the eye using a micropipette
or capillary tube and measuring the depression of the freezing
point that results from heightened osmolarity. However, these ex
vivo measurements are often plagued by many difficulties. For
example, to perform freezing point depression analysis of the tear
sample, a relatively large volume must be collected, typically on
the order of 20 microliters (.mu.L) of a tear film. Because no more
than about 10 to 100 nanoliters (nL) of tear sample can be obtained
at any one time from a KCS patient, the collection of sufficient
amounts of fluid for conventional ex vivo techniques requires a
physician to induce reflex tearing in the patient. Reflex tearing
is caused by a sharp or prolonged irritation to the ocular surface,
akin to when a large piece of dirt becomes lodged in one's eye.
Reflex tears are more dilute, i.e. have fewer solute ions than the
tears that am normally found on the eye. Any dilution of the tear
film invalidates the diagnostic ability of an osmolarity test for
dry eye, and therefore make currently available ex vivo methods
prohibitive in a clinical setting.
[0009] A similar ex vivo technique is vapor pressure osmometry,
where a small, circular piece of filter paper is lodged underneath
a patient's eyelid until sufficient fluid is absorbed. The filter
paper disc is placed into a sealed chamber, whereupon a cooled
temperature sensor measures the condensation of vapor on its
surface. Eventually the temperature sensor is raised to the dew
point of the sample. The reduction in dew point proportional to
water is then converted into osmolarity. Because of the induction
of reflex tearing and the large volume requirements for existing
vapor pressure osmometers, they are currently impractical for
determination of dry eye.
[0010] The Clifton Nanoliter Osmometer (available from Clifton
Technical Physics of Hartford, N.Y., USA) has been used extensively
in laboratory settings to quantify the solute concentrations of KCS
patients, but the machine requires a significant amount of training
to operate. It generally requires hour-long calibrations and a
skilled technician in order to generate acceptable data. The
Clifton Nanoliter Osmometer is also bulky and relatively expensive.
These characteristics seriously detract from it use as a clinical
osmometer.
[0011] In contrast to ex vivo techniques that measure osmolarity of
tear samples removed from the ocular surface, an in vivo technique
that attempted to measure osmolarity directly on the ocular surface
used a pair flexible pair of electrodes that were placed directly
underneath the eyelid of the patient. The electrodes were then
plugged into an LCR meter to determine the conductivity of the
fluid surrounding them. While it has long been known that
conductivity is directly related to the ionic concentration, and
hence osmolarity of solutions, placing the sensor under the eyelid
for half a minute likely induced reflex tearing. Furthermore, these
electrodes were difficult to manufacture and posed increased health
risks to the patient as compared to simply collecting tears with a
capillary.
[0012] It should be apparent from the discussion above that current
osmolarity measurement techniques are unavailable in a clinical
setting and can't attain the volumes necessary for dry eye
patients. Thus, there is a need for an improved, clinically
feasible, nanoliter-scale osmolarity measurement. The present
invention satisfies this need. Tears fulfill an essential role in
maintaining ocular surface integrity, protecting against microbial
challenge, and preserving visual acuity. These functions in turn,
are critically dependent upon the composition and stability of the
tear film structure, which includes an underlying mucin foundation,
a middle aqueous component, and an overlying lipid layer.
Disruption, deficiency, or absence of the tear film can severely
impact the eye.
SUMMARY
[0013] In accordance with the invention, a fluid sample is measured
with a tear film measuring system that includes a processing device
that receives a sample chip comprising a sample region configured
to contain an aliquot volume of sample fluid, the processing device
configured to perform analyses of osmolarity and of one or more
biomarkers within the sample fluid, wherein the analysis of
biomarkers includes normalization of biomarker concentration
values. Processing in accordance with the invention includes
receiving an output signal from a sample region of a sample chip
that is configured to produce an osmolarity output signal that
indicates energy properties of an aliquot volume of the sample
fluid, wherein the osmolarity output signal is correlated with
osmolarity of the sample fluid, receiving an output signal from the
sample region of the sample chip that is configured to produce a
biomarker output signal that indicates chemical properties of the
sample fluid, wherein the biomarker output signal is correlated
with biomarker concentration of the sample fluid, processing the
osmolarity output signal to produce an osmolarity value for the
sample fluid and processing the biomarker output signal to produce
a biomarker concentration value for the sample fluid, and
determining an Adjusted Biomarker Level that provides normalization
of biomarker concentration values. The normalization of biomarker
concentration values can correct for patient-specific tear
homeostasis and for clinician induced tear sampling variance in
connection with obtaining the sample fluid. The processing of the
osmolarity output signal and processing the biomarker output signal
can be performed simultaneously or serially.
[0014] These and other features, aspects, and embodiments of the
invention are described below in the section entitled "Detailed
Description."
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Features, aspects, and embodiments of the inventions are
described in conjunction with the attached drawings, in which:
[0016] FIG. 1 illustrates an aliquot-sized sample receiving chip
for measuring the osmolarity of a sample fluid;
[0017] FIG. 2 illustrates an alternative embodiment of a sample
receiving chip that includes a circuit region with an array of
electrodes imprinted with photolithography techniques;
[0018] FIG. 3 illustrates another alternative embodiment of the
FIG. 1 chip, wherein a circuit region includes printed electrodes
arranged in a plurality of concentric circles;
[0019] FIG. 4 is a top view of the chip shown in FIG. 2;
[0020] FIG. 5 is a top view of the chip shown in FIG. 3;
[0021] FIG. 6 is a block diagram of an osmolarity measurement
system configured in accordance with the present invention;
[0022] FIG. 7 is a perspective view of a tear film osmolarity
measurement system constructed in accordance with the present
invention;
[0023] FIG. 8 is a side section of the sample receiving chip
showing the opening in the exterior packaging;
[0024] FIG. 9 is a calibration curve relating the sodium content of
the sample fluid with electrical conductivity;
[0025] FIG. 10 illustrates a hinged base unit of the osmometer that
utilizes the sample receiving chips described in FIGS. 1-5;
[0026] FIG. 11 illustrates a probe card configuration for the
sample receiving chip and processing unit;
[0027] FIG. 12 is a flowchart describing an exemplary osmolarity
measurement technique in accordance with the invention;
[0028] FIG. 13 is a flow chart illustrating a method for
calibrating an osmolarity measuring system in accordance with
another example embodiment of the invention;
[0029] FIG. 14 is a flow chart illustrating a method for
calibrating an osmolarity measuring system in accordance with
another example embodiment of the invention;
[0030] FIG. 15 is a flow chart illustrating a method for
calibrating an osmolarity measuring system in accordance with
another example embodiment of the invention.
[0031] FIG. 16 is an image showing residual salt crystals on a
miroelectrode array;
[0032] FIG. 17 is a graph illustrating a typical response when a
sample fluid is introduced to a microelectrode array;
[0033] FIG. 18 is a graph illustrating a response when a sample
fluid is introduced to a microelectrode array that contains
residual salt; and
[0034] FIG. 19 is a flow chart illustrating a method for
calibrating an osmolarity measuring system in accordance with
another example embodiment of the invention.
[0035] FIG. 20 is a flow chart illustrating biomarker normalization
in accordance with the invention.
[0036] FIG. 21 is a plan view of a receiving substrate in which
osmolarity is multiplexed in space with biomarker detection.
[0037] FIG. 22 is a detail view of the FIG. 21 receiving substrate
showing the arrangement of electrodes in the sample region.
DETAILED DESCRIPTION
[0038] Exemplary embodiments are described for measuring the
osmolarity of an aliquot volume of a sample fluid (e.g., tear film,
sweat, blood, or other fluids). The exemplary embodiments are
configured to be relatively fast, non-invasive, inexpensive, and
easy to use, with minimal injury of risk to the patient. Accurate
measurements can be provided with as little as nanoliter volumes of
a sample fluid. For example, a measuring device configured in
accordance with the invention enables osmolarity measurement with
no more than 200 .mu.L of sample fluid, and typically much smaller
volumes can be successfully measured. In one embodiment described
further below, osmolarity measurement accuracy is not compromised
by variations in the volume of sample fluid collected, so that
osmolarity measurement is substantially independent of collected
volume. The sample fluid can include tear film, sweat, blood, or
other bodily fluids. It should be noted, however, that sample fluid
can comprise other fluids, such as milk or other beverages.
[0039] FIG. 1 illustrates an exemplary embodiment of an osmolarity
chip 100 that can be used to measure the osmolarity of a sample
fluid 102, such as a tear film sample. In the FIG. 1 embodiment,
the chip 100 includes a substrate 104 with a sample region having
sensor electrodes 108, 109 and circuit connections 110 imprinted on
the substrate. The electrodes and circuit connections are
preferably printed using well-known photolithographic techniques.
For example, current techniques enable the electrodes 108, 109 to
have a diameter in the range of approximately one (1) to eighty
(80) microns, and spaced apart sufficiently so that no conductive
path exists in the absence of sample fluid. Currently available
techniques, however, can provide electrodes of less than one micron
in diameter, and these are sufficient for a chip constructed in
accordance with the invention. The amount of sample fluid needed
for measurement is no more than is necessary to extend from one
electrode to the other, thereby providing an operative conductive
path. The photolithographic scale of the chip 100 permits the
measurement to be made for aliquot-sized samples in a micro- or
nano-scale level. For example, reliable osmolarity measurement can
be obtained with a sample volume of less than 20 .mu.L of tear
film. A typical sample volume is less than one hundred nanoliters
(100 nL). It is expected that it will be relatively easy to collect
10 nL of a tear film sample even from patients suffering from dry
eye.
[0040] The chip 100 is configured to transfer energy to the sample
fluid 102 and enable detection of the sample fluid energy
properties. In this regard, a current source is applied across the
electrodes 108, 109 through the connections 110. The osmolarity of
the sample fluid can be measured by sensing the energy transfer
properties of the sample fluid 102. The energy transfer properties
can include, for example, electrical conductivity, such that the
impedance of the sample fluid is measured, given a particular
amount of electrical power (e.g., current) that is transferred into
the sample through the connections 110 and the electrodes 108,
109.
[0041] If conductivity of the sample fluid is to be measured, then
preferably a sinusoidal signal on the order of ten volts at
approximately 100 kHz is applied. The real and imaginary parts of
the complex impedance of the circuit path from one electrode 108
through the sample fluid 102 to the other electrode 109 are
measured. At the frequencies of interest, it is likely that the
majority of the electrical signal will be in the real half of the
complex plane, which reduces to the conductivity of the sample
fluid. This electrical signal (hereafter referred to as
conductivity) can be directly related to the ion concentration of
the sample fluid 102, and the osmolarity can be determined.
Moreover, if the ion concentration of the sample fluid 102 changes,
the electrical conductivity and the osmolarity of the fluid will
change in a corresponding manner. Therefore, the osmolarity is
reliably obtained. In addition, because the impedance value does
not depend on the volume of the sample fluid 102, the osmolarity
measurement can be made substantially independent of the sample
volume.
[0042] As an alternative to the input signal described above, more
complex signals can be applied to the sample fluid whose response
will contribute to a more thorough estimate of osmolarity. For
example, calibration can be achieved by measuring impedances over a
range of frequencies. These impedances can be either simultaneously
(via combined waveform input and Fourier decomposition) or
sequentially measured. The frequency versus impedance data will
provide information about the sample and the relative performance
of the sample fluid measurement circuit.
[0043] FIG. 2 illustrates an alternative embodiment of a sample
receiving chip 200 that measures osmolarity of a sample fluid 202,
wherein the chip comprises a substrate layer 204 with a sample
region 206 comprising an imprinted circuit that includes an array
of electrodes 208. In the illustrated embodiment of FIG. 2, the
sample region 206 has a 5-by-5 array of electrodes that are
imprinted with photolithographic techniques, with each electrode
208 having a connection 210 to one side of the substrate 204. Not
all of the electrodes 208 in FIG. 2 are shown with a connection,
for simplicity of illustration. The electrodes provide measurements
to a separate processing unit, described further below.
[0044] The electrode array of FIG. 2 provides a means to measure
the size of the tear droplet 202 by detecting the extent of
conducting electrodes 208 to thereby determine the extent of the
droplet. In particular, processing circuitry can determine the
number of electrodes that are conducting, and therefore the number
of adjacent electrodes that are covered by the droplet 202 will be
determined. The planar area of the substrate that is covered by the
sample fluid is thereby determined. With a known nominal surface
tension of the sample fluid, the height of the sample fluid volume
over the planar area can be reliably estimated, and therefore the
volume of the droplet 202 can be determined.
[0045] FIG. 3 illustrates another alternative embodiment of a
sample receiving chip 300 on which a sample fluid 302 is deposited.
The chip comprises a substrate layer 304, wherein a sample region
306 is provided with electrodes 308 that are configured in a
plurality of concentric circles. Each electrode 308 can be
connected to one side of substrate layer 304 by connections 310. In
a manner similar to the square array of FIG. 2, the circular
arrangement of the FIG. 3 electrodes 308 also provides an estimate
of the size of the sample fluid volume 302 because the droplet
typically covers a circular or oval area of the sample region 302.
Processing circuitry can detect the largest (outermost) circle of
electrodes that are conducting, and thereby determine a planar area
of coverage by the fluid sample. As before, the determined planar
area provides a volume estimate, in conjunction with a known
surface tension and corresponding volume height of the sample fluid
302. In the FIG. 3 illustrated embodiment, the electrodes 308 can
be printed using well known photolithography techniques that
currently permit electrodes to have a diameter in the range of one
(1) to eighty (80) microns. This allows the submicroliter droplet
to substantially cover the electrodes. The electrodes can be
printed over an area sized to receive the sample fluid, generally
covering 1 mm.sup.2 to 1 cm.sup.2.
[0046] The electrodes and connections shown in FIG. 1, FIG. 2, and
FIG. 3 can be imprinted on the respective substrate layers as
electrodes with contact pads, using photolithographic techniques.
For example, the electrodes can be formed with different conductive
metalization such as aluminum, platinum, titanium,
titanium-tungsten, and other similar material. In one embodiment,
the electrodes can be formed with a dielectric rim to protect field
densities at the edges of the electrodes. This can reduce an
otherwise unstable electric field at the rim of the electrode.
[0047] Top views of the exemplary embodiments of the chips 200 and
300 are illustrated in FIG. 4 and FIG. 5, respectively. The
embodiments show the detailed layout of the electrodes and the
connections, and illustrate how each electrode can be electrically
connected for measuring the electrical properties of a sample
droplet. As mentioned above, the layout of the electrodes and the
connections can be imprinted on the substrate 100, 200, 300 using
well-known photolithographic techniques.
[0048] FIG. 6 is a block diagram of an osmometry system 600
configured in accordance with an embodiment of the present
invention, showing how information is determined and used in a
process that determines osmolarity of a sample fluid. The osmometry
system 600 includes a measurement device 604 and a processing
device 606. The measurement device receives a volume of sample
fluid from a collection device 608. The collection device can
comprise, for example, a micropipette or capillary tube. The
collection device 608 collects a sample tear film of a patient,
such as by using negative pressure from a fixed-volume micropipette
or charge attraction from a capillary tube to draw a small tear
volume from the vicinity of the ocular surface of a patient.
[0049] The measurement device 604 can comprise a system that
transfers energy to the fluid in the sample region and detects the
imparted energy. For example, the measurement device 604 can
comprise circuitry that provides electrical energy in a specified
waveform (such as from a function generator) to the electrical path
comprising two electrodes bridged by the sample fluid. The
processing device 606 detects the energy imparted to the sample
fluid and determines osmolarity. The processing device can
comprise, for example, a system including an RLC multimeter that
produces data relating to the reactance of the fluid that forms the
conductive path between two electrodes, and including a processor
that determines osmolarity through a table look-up scheme. If
desired, the processing device can be housed in a base unit that
receives one of the chips described above.
[0050] As mentioned above, a sample sufficient to provide an
osmolarity measurement can contain less than 20 microliters (.mu.L)
of fluid. A typical sample of tear film in accordance with the
invention is collected by a fluid collector such as a capillary
tube, which often contains less than one microliter of tear film.
Medical professionals will be familiar with the use of
micropipettes and capillary tubes, and will be able to easily
collect the small sample volumes described herein, even in the case
of dry eye sufferers.
[0051] The collected sample fluid is expelled from the collection
device 608 to the measurement device 604. The collection device can
be positioned above the sample region of the chip substrate either
manually by a medical professional or by being mechanically guided
over the sample region. In one embodiment, for example, the
collection device (e.g., a capillary tube) is mechanically guided
into position with an injection-molded plastic hole in a base unit,
or is fitted to a set of clamps with precision screws (e.g., a
micromanipulator with needles for microchip interfaces). In another
embodiment, the guide is a computer-guided feedback control
circuitry that holds the capillary tube and automatically lowers it
into the proper position.
[0052] The electrodes and connections of the chips measure energy
properties of the sample fluid, such as conductivity, and enable
the measured properties to received by the processing device 606.
The measured energy properties of the sample fluid include
electrical conductivity and can also include other parameters, such
as both parts of the complex impedance of the sample, the variance
of the noise in the output signal, and the measurement drift due to
resistive heating of the sample fluid. The measured energy
properties are processed in the processing device 606 to provide
the osmolarity of the sample. In one embodiment, the processing
device 606 comprises a base unit that can accept a chip and can
provide electrical connection between the chip and the processing
device 606. In another embodiment, the base unit can include a
display unit for displaying osmolarity values. It should be noted
that the processing device 606 and, in particular, the base unit
can be a hand-held unit.
[0053] FIG. 7 is a perspective view of a tear film osmolarity
measuring system 700 constructed in accordance with the present
invention. In the illustrated embodiment of FIG. 7, the exemplary
system 700 includes a measuring unit 701 that comprises a chip,
such as one of the chips described above, and a connector or socket
base 710, which provides the appropriate measurement output. The
system 700 determines osmolarity by measuring electrical
conductivity of the sample fluid: Therefore, the measurement chip
701 comprises a semiconductor integrated circuit (IC) chip with a
substrate having a construction similar to that of the chips
described above in connection with FIG. 1 through FIG. 5. Thus, the
chip 701 includes a substrate layer with a sample region that is
defined by at least two electrodes printed onto the substrate layer
(such details are of a scale too small to be visible in FIG. 7; see
FIG. 1 through FIG. 5). The substrate and sample region are encased
within an inert package, in a manner that will be known to those
skilled in the art. In particular, the chip 701 is fabricated using
conventional semiconductor fabrication techniques into an IC
package 707 that includes electrical connection legs 708 that
permit electrical signals to be received by the chip 701 and output
to be communicated outside of the chip. The packaging 707 provides
a casing that makes handling of the chip more convenient and helps
reduce evaporation of the sample fluid.
[0054] FIG. 8 shows that the measurement chip 701 is fabricated
with an exterior opening hole 720 into which the sample fluid 702
is inserted. Thus, the hole 720 can be formed in the semiconductor
packaging 707 to provide a path through the chip exterior to the
substrate 804 and the sample region 806. The collection device
(such as a micropipette or capillary tube) 808 is positioned into
the hole 720 such that the sample fluid 702 is expelled from the
collection device directly onto the sample region 806 of the
substrate 804. The hole 720 is sized to receive the tip of the
collection device. The hole 720 forms an opening or funnel that
leads from the exterior of the chip onto the sample region 806 of
the substrate 804. In this way, the sample fluid 702 is expelled
from the collection device 808 and is deposited directly on the
sample region 806 of the substrate 804. The sample region is sized
to receive the volume of sample fluid from the collection device.
In FIG. 8, for example, the electrodes form a sample region 806
that is generally in a range of approximately 1 mm.sup.2 to 1
cm.sup.2 in area.
[0055] Returning to FIG. 7, the chip 701 can include processing
circuitry 704 that comprises, for example, a function generator
that generates a signal of a desired waveform, which is applied to
the sample region electrodes of the chip, and a voltage measuring
device to measure the root-mean-square (RMS) voltage value that is
read from the chip electrodes. The function generator can produce
high frequency alternating current (AC) to avoid undesirable direct
current (DC) effects for the measurement process. The voltage
measuring device can incorporate the functionality of an RLC
measuring device. Thus, the chip 701 can incorporate the
measurement circuitry as well as the sample region electrodes. The
processing circuitry can include a central processing unit (CPU)
and associated memory that can store programming instructions (such
as firmware) and also can store data. In this way, a single chip
can include the electrodes and associated connections for the
sample region, and on a separate region of the chip, can also
include the measurement circuitry. This configuration will minimize
the associated stray resistances of the circuit structures.
[0056] As noted above, the processing circuitry 70 applies a signal
waveform to the sample region electrodes. The processing circuitry
also receives the energy property signals from the electrodes and
determines the osmolarity value of the sample fluid. For example,
the processing unit receives electrical conductivity values from a
set of electrode pairs. Those skilled in the art will be familiar
with techniques and circuitry for determining the conductivity of a
sample fluid that forms a conducting path between two or more
electrodes.
[0057] In the FIG. 7 embodiment, the processing unit 704 produces
signal waveforms at a single frequency, such as 100 kHz and 10
Volts peak-to-peak. The processing circuitry 704 then determines
the osmolarity value from the sodium content correlated to the
electrical conductivity using a calibration curve, such as the
curve shown in FIG. 9. In this case, the calibration curve is
constructed as a transfer function between the electrical
conductivity (voltage) and the osmolarity value (i.e., the sodium
content). It should be noted, however, that other calibration
curves can also be constructed to provide transfer functions
between other energy properties and the osmolarity value. For
example, the variance, autocorrelation and drift of the signal can
be included in an osmolarity calculation. If desired, the
osmolarity value can also be built upon multi-variable correlation
coefficient charts or neural network interpretation so that the
osmolarity value can be optimized with an arbitrarily large set of
measured variables.
[0058] In an alternate form of the FIG. 7 embodiment, the
processing unit 704 produces signal waveforms of a predetermined
frequency sweep, such as 1 kHz to 100 kHz in 1 kHz increments, and
stores the conductivity and variance values received from the set
of electrode pairs at each frequency. The output signal versus
frequency curve can then be used to provide higher order
information about the sample which can be used with the
aforementioned transfer functions to produce an ideal osmolarity
reading.
[0059] As shown in FIG. 7, the base socket connector 710 receives
the pins 708 of the chip 701 into corresponding sockets 711. The
connector 710, for example, can supply the requisite electrical
power to the processing circuitry 704 and electrodes of the chip.
Thus, the chip 701 can include the sample region electrodes and the
signal generator and processing circuitry necessary for determining
osmolarity, and the output comprising the osmolarity value can be
communicated off the chip via the pins 708 through the connector
710 and to a display readout.
[0060] If desired, the base connector socket 710 can include a
Peltier layer 712 located beneath the sockets that receive the pins
708 of the chip 701. Those skilled in the art will understand that
a Peltier layer comprises an electrical/ceramic junction such that
properly applied current can cool or heat the Peltier layer. In
this way, the sample chip 701 can be heated or cooled, thereby
further controlling evaporation of the sample fluid. It should be
apparent that evaporation of the sample fluid should be carefully
controlled, to ensure accurate osmolarity values obtained from the
sample fluid.
[0061] FIG. 10 shows an alternative embodiment of an osmometer in
which the chip does not include an on-chip processing unit such as
described above, but rather includes limited circuitry comprising
primarily the sample region electrodes and interconnections. That
is, the processing unit is separately located from the chip and can
be provided in the base unit.
[0062] FIG. 10 shows in detail an osmometer 1000 that includes a
base unit 1004, which houses the base connector 710, and a hinged
cover 1006 that closes over the base connector 710 and a received
measurement chip 701. Thus, after the sample fluid has been
dispensed on the chip, the chip is inserted into the socket
connector 710 of the base unit 1004 and the hinged cover 1006 is
closed over the chip to reduce the rate of evaporation of the
sample fluid.
[0063] It should be noted that the problem with relatively fast
evaporation of the sample fluid can generally be handled in one of
two ways. One way is to measure the sample fluid voltage quickly as
soon possible after the droplet is placed on the sample region of
the chip. Another way is to enable the measuring unit to measure
the rate of evaporation along with the corresponding changes in
conductivity values. The processing unit can then post-process the
output to estimate the osmolarity value. The processing can be
performed in the hardware or in software stored in the hardware.
Thus, the processing unit can incorporate different processing
techniques such as using neural networks to collect and learn about
characteristic of the fluid samples being measured for osmolarity,
as well as temperature variations, volume changes, and other
related parameters so that the system can be trained in accordance
with neural network techniques to make faster and more accurate
osmolarity measurements.
[0064] FIG. 11 shows another alternative construction, in which the
osmolarity system utilizes a sample receiving chip 1102 that does
not include IC packaging such as shown in FIG. 7. Rather, the FIG.
11 measurement chip 1102 is configured as a chip with an exposed
sample region comprising the electrodes and associated connections,
but the processing circuitry is located in the base unit for
measuring the energy properties of the sample fluid. In this
alternative construction, a connector similar to the connector
socket 710 allows transmission of measured energy properties to the
processing unit in the base unit. Those skilled in the art will
understand that such a configuration is commonly referred to a
probe card structure.
[0065] FIG. 11 shows a probe card base unit 1100 that receives a
sample chip probe card 1102 that comprises a substrate 1104 with a
sample region 1106 on which are formed electrodes 1108 that are
wire bonded to edge connectors 1110 of the probe card. When the
hinged lid 1112 of the base unit is closed down over the probe
card, connecting tines 1114 on the underside of the lid come into
mating contact with the edge connectors 1110. In this way, the
electrodes of the sample region 1106 are coupled to the processing
circuitry and measurement can take place. The processing circuitry
of the probe card embodiment of FIG. 11 can be configured in either
of the configurations described above. That is, the processing to
apply current to the electrodes and to detect energy properties of
the sample fluid and determine osmolarity can be located on-chip,
on the substrate of the probe card 1102, or the processing
circuitry can be located off-chip, in the base unit 1100.
[0066] In all the alternative embodiments described above, the
osmometer is used by placing a new measurement chip into the base
unit while the hinged top is open. Upon placement into the base
unit the chip is lowered up and begins monitoring its environment.
Recording output signals from the chip at a rate of, for example, 1
kHz, will fully capture the behavior of the system. Placing a
sample onto any portion of the electrode array generates high
signal-to-noise increase in conductivity between ally pair of
electrodes covered by the sample fluid. The processing unit will
recognize the change in conductivity as being directly related to
the addition of sample fluid, and will begin conversion of
electronic signals into osmolarity data once this type of change is
identified. This strategy occurs without intervention by medical
professionals. That is, the chip processing is initiated upon
coupling to the base unit and is not dependent on operating the lid
of the base unit or any other user intervention.
[0067] In any of the configurations described above, either the
"smart chip" with processing circuitry on-chip (FIG. 7), or the
electrode-only configuration with processing circuitry off-chip
(FIG. 10), in a packaged chip (FIG. 7 and FIG. 10) or in a probe
card (FIG. 1), the sample receiving chip can be disposed of after
each use, so that the base unit serves as a platform for
interfacing with the disposable measurement chip. As noted, the
base unit can also include relevant control, communication, and
display circuits (not shown), as well as software, or such features
can be provided off-chip in the base unit. In this regard, the
processing circuitry can be configured to automatically provide
sufficient power to the sample region electrodes to irreversibly
oxidize them after a measurement cycle, such that the electrodes
are rendered inoperable for any subsequent measurement cycle. Upon
inserted a used chip into the base unit, the user will be given an
indication that the electrodes are inoperable. This helps prevent
inadvertent multiple use of a sample chip, which can lead to
inaccurate osmolarity readings and potentially unsanitary
conditions.
[0068] A secondary approach to ensure that a previously used chip
is not placed back into the machine includes encoding serial
numbers, or codes directly onto the chip. The base unit will store
the used chip numbers in memory and cross-reference them against
new chips placed in the base connector. If the base unit finds that
the serial number of the used chip is the same as an old chip, then
the system will refuse to measure osmolarity until a new chip is
inserted. It is important to ensure use of a new chip for each test
because proteins adsorb and salt crystals form on the electrodes
after evaporation has run its course, which corrupt the integrity
of the measuring electrodes.
[0069] FIG. 12 is a flowchart describing an exemplary (osmolarity
measurement technique in accordance with the invention. A body
fluid sample, such as a tear film, is collected at box 1300. The
sample typically contains less than one microliter. At box 1302,
the collected sample is deposited on a sample region of the chip
substrate. The energy properties of the sample are then measured at
box 1304. The measured energy properties are then processed, at box
1306, to determine the osmolarity of the sample. If the chip
operates in accordance with electrical conductivity measurement,
then the measurement processing at box 1306 can include the
"electrode oxidation" operation described above that renders the
chip electrodes inoperable for any subsequent measuring cycles.
[0070] In the measurement process for a conductivity measuring
system, a substantially instantaneous shift is observed from the
open circuit voltage to a value that closely represents the state
of the sample at the time of collection, upon placement of a sample
tear film on an electrode array of the substrate. Subsequently, a
drift in the conductivity of the sample will be reflected as a
continual change in the output.
[0071] The output of the measurement chip can be a time-varying
voltage that is translated into an osmolarity value. Thus, in a
conductivity-based system, more information than just the
"electrical conductivity" of the sample can be obtained by
measuring the frequency response over a wide range of input
signals, which improves the end stage processing. For example, the
calibration can be made over a multiple frequencies (e.g., measure
ratio of signals at 10, 20, 30, 40, 50, 100 Hz) to make the
measurement process a relative calculation. This makes the
chip-to-chip voltage drift small. The standard method for
macroscale electrode based measurements (i.e. in a pH meter, or
microcapillary technique) is to rely upon known buffers to set up a
linear calibration curve. Because photolithography is a relatively
reproducible manufacturing technique, when coupled to a frequency
sweep, calibration can be performed without operator
intervention.
[0072] As mentioned above, the processing of the energy properties
can be performed in a neural network configuration, where the
seemingly disparate measured data points obtained from the energy
properties can be used to provide more accurate osmolarity reading
than from a single energy property measurement. For example, if
only the electrical conductivity of the sample is measured, then
the calibration curve can be used to simply obtain the osmolarity
value corresponding to the conductivity. This osmolarity value,
however, generally will not be as accurate as the output of the
neural network.
[0073] The neural network can be designed to operate on a
collection of calibration curves that reflects a substantially
optimized transfer function between the energy properties of the
sample fluid and the osmolarity. Thus, in one embodiment, the
neural network constructs a collection of calibration curves for
all variables of interest, such as voltage, evaporation rate and
volume change. The neural network can also construct or receive as
an input a priority list that assigns an importance factor to each
variable to indicate the importance of the variable to the final
outcome, or the osmolarity value. The neural network constructs the
calibration curves by training on examples of real data where the
final outcome is known a priori. Accordingly, the neural network
will be trained to predict the final outcome from the best possible
combination of variables. This neural network configuration that
processes the variables in an efficient combination is then loaded
into the processing unit residing in the measurement chip 701 or
the base unit. Once trained, the neural network can be configured
in software or hardware.
[0074] The ability to identify and subtract out manufacturing
defects in the electrodes prior to osmolarity testing can also be
important. This too can be accomplished via calibration of an
osmolarity calibration device that comprises an osmolarity chip,
such as chip 1200 illustrated in FIG. 2. This type of calibration
can also be achieved, possibly in a more efficient manner, through
the use of neural networks, but it will be understood that such
networks are not necessary to achieve the calibration processes
described in the following description.
[0075] Classically, bare metal electrodes were considered poor
measuring devices when placed in direct contact with an
electrochemical solution of interest. Foremost, there can exist a
double layer of counter ions that surround the electrode at the
metal/solution interface that can impose a field of sufficient
magnitude to significantly alter the ion quantity of interest. In
bulk solutions, convection currents or stirring can disrupt these
distributions and cause time varying noise, whose magnitude is on
the order of the relevant signal. Further, the polarizability and
hysterisis of the electrodes can cause problems if sourcing small
signals to the electrodes. Finally, large DC or low frequency AC
sources from the electrode can also cause irreversible electrolysis
that results in bubbling and oxidation of important biological
species. Bubbles introduce variable dielectric shifts near the
electrode and invalidate inferences drawn about the solution from
voltages measured under such conditions.
[0076] A conventional solution for these effects is to physically
separate the electrodes from the solution through a salt bridge,
whereupon bubbling and other nonlinearities in the immediate
vicinity of the active electrodes are largely irrelevant to the
steady state distribution of ions that flow far from the
electrodes. As an example, in devices configured to measure the pH
of a solution, the metal electrodes can be separated from the bulk
solution with a semipermeable membrane such as glass or ceramic
material. Moreover, the metal electrode within the semipermeable
membrane is generally comprised of Ag (silver) or calomel (mercury)
immersed in a silver chloride or mercurous chloride solution. This
allows a single chemical reaction to dominate action close to the
electrode. The reaction can be kept close to equilibrium, and when
a gradient of ions is created across the semipermeable membrane, an
osmotic force is transmitted to the electrode surface through the
symmetric redox reaction which drives the system back towards
equilibrium. In this way, ions are balanced at the glass-solution
and electrode-buffer interface, and nonlinearities can be
minimized.
[0077] In contrast to typical measurement systems, however,
clinical measurements of human tear film osmolarity require far
smaller electrodes than traditional systems. This is due to the
fact that tens of nanoliters represent the maximum viable
collection volume from patients with, e.g., keratoconjunctivitis
sicca. As described above, the systems and methods described herein
can allow for a clinical device for tear measurements that can meet
the strict requirements for accurate measurements and diagnosis in
this area by using bare metal electrodes printed on a microchip,
e.g., as shown in FIG. 1 and FIG. 2. As a result, none of the
traditional solutions to electrode shielding are feasible for such
devices because the physical dimensions are far too small. At, for
example, 80 .mu.m in diameter, the photolithographed electrodes
preclude membranes from being manufactured in a cost effective
manner. For example, a spin coated gel permeation layer is
prohibitively expensive, results in a low yield process, and
introduces several manufacturing variances. Further, osmotic
perturbations due to salt bridge gradients would overwhelm the
minuscule sample volume of interest. Therefore, many of the typical
methods for taking measurements with macroscale electrodes cannot
be applied to microelectrodes, and additional issues of calibration
remain.
[0078] In order to establish a linear calibration profile, where
input directly scales with output, conventional macroscale
electrodes are typically immersed in multiple known standards. For
instance, pH meters will use a set of three buffers at pH 4, 7 and
10, with each fluid marking a point for the fitted line. Between
each calibration point, the macroscale electrode can be washed and
dried to ensure that the standards do not mix. If one assumes that
the electrode buffer inside the glass chamber is of a certain
concentration, then calibration can be performed with as little as
one standard point. Over time however, the once homogeneous
electrode buffer becomes contaminated with the substances it has
measured, which then requires at least a two-point calibration in
order to be precise.
[0079] When working with microelectrodes, however, conventional
calibration steps, such as those described above, are often
impossible to perform without risking damage to the array and
compromising any ensuing measurements. For instance after a
calibration standard has been placed on the chip, it is impractical
to clean the array with paper, because scratches on the electrode
surface will result in exceedingly high current densities at the
scratch edge, which then leads to bubbling and invalid
measurements. Furthermore, if one were to use a model of human
tears for the calibration standard, i.e. with 10 mg/ml BSA as a
constituent, protein adsorption to the electrode surface would
corrupt the purity of a clinical measurement. Finally, if a small
amount of salt solution was used to set calibration points, the
fluid would evaporate, leaving a very noticeable salt crystal upon
random parts of the chip surface. This residual salt will then
dissolve into any subsequent sample that is placed on the chip, and
unlike the volume independence displayed by conductivity, slight
differences in the amount of fluid deposited as a standard will
result in different amounts of salt added to the fluid sample of
interest.
[0080] Ultimately, a clinical test for dry eye requires a
conversion from the relative motion of ions in solution to an
absolute osmolarity that can be compared between tests over time.
The final value must be independent of the measuring device and
stable over time to qualify for diagnostic purposes. Accordingly,
the ability to calibrate a microelectrode array, such as those
described above, can be hampered by several remaining challenges
when attempting to obtain the strictest possible tolerances for the
measuring device.
[0081] As described below, however, several approaches can be used
to calibrate a microelectrode array, such as those described above,
in accordance with the systems and methods described herein. These
approaches can each start by determining an intrinsic conductivity
for each electrode in the array. This intrinsic conductivity can
then be stored and used to subtract out the effect of the intrinsic
conductivity form final measurements of the electrical properties
of a test fluid, such as a tear. Depending on the embodiment, a
standard may or may not be used in determining a calibration factor
for the electrodes. Further, when a standard is used, a subsequent
washing step may or may not be included.
[0082] It should also be pointed out that the various approaches
can be combined in a modular fashion to produce ever more accurate
calibration results. The approaches can thus be used tiered to
produce successive levels of intricacy in order to minimize
variability between tests.
[0083] FIG. 13 is a flow chart that illustrates one embodiment of a
method for calibrating an osmolarity measuring device that does not
use a standard in accordance with one embodiment of the systems and
methods described herein. At box 1402, the intrinsic conductivity
of the electrodes is measured. The measured intrinsic conductivity
for each electrode can then be stored on a memory. At box 1404, a
sample fluid of interest, such as a tear film, is introduced to the
measuring device, and the electrical properties of the sample fluid
are measured at box 1406. In one embodiment, the processing
circuitry also identifies the electrodes from the electrode array
that are in contact with the sample fluid at box 1408. The
electrodes that are in contact with the sample fluid are conducting
electrodes, and the identity of the conducting electrodes can also
be stored in memory. At box 1410, the processing circuitry adjusts
the measured electrical properties of the sample fluid to adjust
for the intrinsic conductivity of the electrodes, on a pair-wise
basis, that performed the measurement of the sample. This
adjustment results an osmolarity measurement of the sample alone,
and is independent of variances in the thickness of electrode
metalization, dielectric deposition, and other variances in the
electrodes that can occur during manufacturing of the measuring
device.
[0084] The intrinsic conductivity can be determined (box 1402), in
one embodiment, by applying a DC current to the electrode array and
measuring the resulting output voltage for each electrode. The
corresponding resistance can then be calculated based on the DC
current and the output voltage and, e.g., stored in memory. In an
alternative embodiment, a more complex signal, e.g., a sine wave,
can be generated in the time domain and applied to the array of
electrodes. The corresponding outputs can then be measured and
stored. A Fourier transform can then be applied to the stored
output data. The result is a map of amplitude versus frequency that
indicates the relative conductance over a range of frequencies for
each electrode. This map can be generated for a range of
frequencies of interest for a particular implementation, e.g., from
the low kHz to the MHz range.
[0085] In order to deliver a current signal to each electrode and
measure the resulting output for calibration purposes, two leads
can be provided for each electrode. The current signal can then be
applied and the output measured, for a given electrode, via the two
leads.
[0086] In such an embodiment, the slope of the resulting
calibration curve can be assumed to be constant over time. The
curve can then be built into a osmolarity measurement device, such
as those described above. Adjustments to the osmolarity
determinations can then be made through electrode resistance
subtraction, which will simply shift the input mapping along the
x-axis of the calibration curve. Other effects, such as humidity
and ambient temperature effects can then, depending on the
embodiment, be accounted for in subsequent signal processing.
[0087] The ability to map out the intrinsic conductivity of each
electrode pair prior to testing also gives a confidence bound to
the array locations. In this manner, the electrode is defined as a
random process of gaussian random variables with a sample mean and
variance as defined by the conductivity calculations above. Any
electrode outside the 95th percentile of the expected variance can
be considered flawed, and its signals can be neglected in future
calculations. This ability to selectively address electrode pairs
in an array enhances the ability to calibrate the reading and
protect against spurious manufacturing defects.
[0088] As an example, it should be remembered that the electrode
array of FIG. 2 provides a means to measure the size of, e.g., a
tear droplet 202 by detecting the extent of conducting electrodes
208 to thereby determine the extent of the droplet. In particular,
processing circuitry can determine the number of electrodes that
are conducting, and therefore the number of adjacent electrodes
that are covered by the droplet 202. The identities of the
electrodes from the array that are conducting and in contact with
the sample fluid 202 can then stored in memory.
[0089] Thus, following the completion of the sample testing, the
intrinsic conductivity of all of the conducting electrode pairs can
be subtracted from the sample output signal to calculate an
osmolarity value indicative of the sample alone. In one embodiment,
it is important to recognize that the sample fluid 202 will not
cover all electrodes in the array. Therefore, only those electrodes
that are conducting and in contact with the sample fluid 202 are
included in the calculation to adjust the sample measurement. As
mentioned, the resulting osmolarity measurement of the sample fluid
202 is therefore made independent of variances in the thickness of
electrode metalization, dielectric deposition, and other variances
that may occur during manufacturing for the conducting electrodes
that perform the measurement.
[0090] While the systems and methods from calibration just
described are useful and simple to implement, requiring minimal
software post processing to accomplish any needed correction, it is
unlikely that this method will detect sharp deformities in
electrode geometries such as metal peaks or rough edges because
these defects will not significantly alter the intrinsic
conductivity of the electrodes. It can be shown that the bare metal
electrodes described above suffice for measurement when high
frequency sine waves are used as input signals to the
microelectrodes. Even at 10 V peak to peak, 10-100 kHz waves do not
initiate bubbling in the aliquot of tear film sample that is being
measured. This is can be due to the fact that within this frequency
range, there is a balance between water polarizability and ionic
mobility, resulting in oscillations of ions rather than bulk
movement. This solves many problems with electrolysis and other DC
electrode problems. However, when a sample fluid is applied,
electrode geometries such as metal peaks or rough edges may cause
bubbling and mar the measurement. Therefore, in order to account
for these effects, it is useful to begin each test with a one- or
two-points standard calibration prior to use.
[0091] FIG. 14 is a flow chart that illustrates one embodiment of a
method for calibrating an osmolarity measuring device with a
standard fluid. At box 1502, a calibration curve is provided on a
memory, and the calibration curve is assumed to be a straight line.
One point of the line is obtained through the assumption that when
the measured electrical properties of the standard are equal to
zero, the osmolarity of the standard is equal to zero. The
electrical properties (i.e. sine wave Fourier transform, etc.) of
the high end of the concentration range, around 500 mOsms, can then
be predefined in memory based on known electrical properties for a
fluid having a known concentration.
[0092] At box 1504, a standard fluid can be deposited onto the
microelectrode array of a measuring device, and the electrical
properties of the standard can be measured at box 1504. The methods
for measuring the electrical properties of the standard fluid can
include the methods described above for measuring the electrical
properties of a sample fluid. A processing device can then be
configured to correlate the measured electrical properties to an
osmolarity value and, e.g., store the osmolarity measurement of the
standard fluid in memory.
[0093] In one embodiment, the standard fluid that is added at box
1504 comprises a small aliquot, for example, 1 .mu.L, of deionized
water. The osmolarity measurement for deionized water can be
registered as a lower bound on the calibration curve since
deionized water exhibits a minimum amount of osmotic character. In
one embodiment, a one-point calibration is used such that the
entire range for the measurement scale of the device can be
extrapolated based on the difference between the expected
osmolarity of deionized water and the actual measured osmolarity of
the standard. At box 1506, the processing device determines a
calibration factor to adjust the slope of the measurement scale to
match the calibration curve. Further, any adjustments to the slope
of the measurement scale are made with the measured fluid per
electrode pair, such that the final value from each electrode pair
is equivalent with all others. The final calibration factor can
then be stored in memory.
[0094] After calibration has been determined, the standard can be
allowed to evaporate from the microelectrode array at box 1508.
Evaporation can be necessary to prevent the standard from mixing
with and corrupting the sample fluid. Deionized water provides an
exemplary standard when the deionized water has such low salt
content that there is no salt crystal deposited on the chip after
evaporation.
[0095] In one embodiment where there is no salt crystal remaining
after the standard evaporates, the sample fluid to be tested can
then be deposited on the microelectrode array of the measuring
device at box 1510. The microelectrode array transfers energy to
the sample fluid and enables the detection of the sample fluid's
electrical properties, which are mapped to an osmolarity
measurement at box 1512 as described above. At box 1514, a
processing device can be configured to adjust the osmolarity
measurement based on the previously determined calibration factor.
The use of the calibration factor results in an osmolarity
measurement that is substantially independent from variances in the
geometry of the microelectrode array.
[0096] The process of FIG. 14 can also be combined with the simpler
process of FIG. 13 in order to improve accuracy.
[0097] FIG. 15 is a flow chart that illustrates another embodiment
of a method for calibrating an osmolarity measuring device using a
standard fluid that is a slat solution. At box 1602, a calibration
curve can be provided on a memory, and the calibration curve can be
assumed to be a straight line. One point of the line is obtained
through the assumption that when the measured electrical properties
of the standard are equal to zero, the osmolarity of the standard
is equal to zero. The electrical properties of the high end of the
concentration range, around 500 mOsms, can be predefined in memory
based on known electrical properties for a fluid having a known
concentration.
[0098] At box 1604 a standard fluid can then be deposited onto the
microelectrode array of a measuring device, and the electrical
properties of the standard can be measured. The methods for
measuring the electrical properties of the standard can, for
example, include the methods described above for measuring the
electrical properties of a sample fluid. A processing device can
then be configured to correlate the measured electrical properties
to an osmolarity value, and store the osmolarity measurement of the
standard on a memory.
[0099] At box 1606, the processing device can be configured to then
determine a calibration factor to adjust the slope of the
measurement scale to match the calibration curve. Further, any
adjustments to the slope of the measurement scale can be made with
the measured fluid on a per electrode pair basis, such that the
final value from each electrode pair is equivalent with all others.
The final calibration factor can then be stored in memory.
[0100] In this process, however, the standard can be a simple salt
solution (NaCl), or a complex salt solution, e.g., with sodium,
potassium, calcium and magnesium salts in physiological ratios.
However, when the salt solution evaporates at box 1608, a very
noticeable salt crystal will often remain on the chip surface as
shown in FIG. 16. When this occurs, the left over salt crystal
should be accounted for in the subsequent osmolarity measurement
that is made at box 1612.
[0101] For example, FIG. 17 demonstrates a typical response when a
sample fluid is introduced to a microelectrode array that does not
include residual salt. In comparison, FIG. 18 shows the response
when residual salt is present on the microelectrode array at the
time the sample fluid is introduced. The presence of a salt crystal
clearly alters the response, such that it steadily declines for a
period before righting itself and heading into the steady
evaporation mode. As shown in FIG. 18, the normal second order
dynamics are suppressed. This is due to the fact that upon sample
placement, the residual salt crystal will begin to dissolve into
the sample fluid. The concentration of residual salt near the
electrode will continue to decrease until its contribution has
become uniformly mixed throughout the sample, whereupon the curve
will begin to rise again due to evaporation.
[0102] During this transient response, dissolving ions between two
measuring electrodes will present a much higher conductivity than
in the originally deposited solution FIG. 16 also shows how a
misplaced drop of salt solution can differentially cover the array
surface, which means that the signal between pairs of electrodes
will be vastly different depending on their proximity to the salt
crystal.
[0103] Therefore, in another embodiment of the systems and methods
for calibrating an osmolarity measuring device, a processing device
can be configured to mathematically eliminate the effects of any
residual salt crystals from the osmolarity measurement of the
sample at box 1614. The effects of the residual salt crystal can,
for example, be eliminated by integrating the descending curves
from every electrode pair, which estimates the amount of salt added
to the solution. The estimation of the amount of salt that is added
is accomplished by summing only the area above the steady state
line, which is determined by a linear regression far from the time
point of sample delivery. These effects are then subtracted out
from the total volume of the sample. As previously discussed, the
total volume of the sample can be estimated by the processing
device based on the number of electrodes that are in contact with
the sample.
[0104] Based on these parameters, the measured concentration of the
sample is adjusted directly. The concentration of the sample is
based on the number of ions per unit of volume. The osmolarity
measurement provides the total number of ions from the sample plus
the residual salt crystal, and the processing device estimates the
volume of the sample. Accordingly, the adjustment requires the
subtraction of the number residual salt ions from the measured
number of total ions in the sample. The number of residual salt
ions is determined through the integration method discussed above.
This method enables the use of a salt solution standard on the
microscale without the need for expensive washing hardware. After
the effect from the residual salt has been subtracted, the
processing device adjusts the resulting osmolarity measurement
based on the previously determined calibration factor at box 1514.
The use of the calibration factor results in an osmolarity
measurement that is substantially independent from variances in the
geometry of the microelectrode array.
[0105] FIG. 19 is a flow chart that illustrates still another
embodiment of a method for calibrating an osmolarity measuring
device with a standard fluid in accordance with the systems and
methods described herein. In the embodiment of FIG. 19, a wash can
be used in conjunction with the application of a standard fluid.
The steps performed at boxes 1902, 1904, and 1906 have been
previously discussed and result in the determination of a
calibration factor based on the measurement of one or more
standards. In one embodiment, the standard contains a simple salt
solution (NaCl), or a complex salt solution, with sodium,
potassium, calcium and magnesium salts in physiological
ratios).
[0106] At box 1908, an action is performed to remove the standard
from the chip before the standard evaporates and prevent the
accumulation of residual salt on the chip. In one embodiment, the
washing step uses a microfluidic chamber attached in series to the
sample receiving substrate to allow a steady stream of deionized
water to flow across the chip surface. Once a standard aliquot has
been deposited, either through a perpendicular microfluidic flow
channel or by manual methods, and the (calibration measurement has
been made, the washing apparatus will flow deionized water across
the electrode surface until the conductivity has reached a steady
state commensurate with the expected deionized water levels. The
steady state conductivity indicates that the chip surface has been
cleaned of any standard and is ready to accept a sample. The flow
is halted and the deionized water is allowed to evaporate on the
chip surface, ideally leaving no salt crystal behind.
[0107] In another embodiment, a valved high pressure air supply can
be implemented to remove the standard. The tube is connected to the
air supply and placed in close proximity to the receiving substrate
and at an acute angle from the surface. The angle is such that a
quick puff of air from the tube forces any fluid from the surface
of the chip to be cleared completely from the substrate. The flow
of air is triggered upon completion of the calibration measurement.
The resulting air flow may be pulsed several times until the signal
at each electrode pair has returned to open circuit values. In
another embodiment, air supply is combined with the microfluidic
washing stage to eliminate the need to evaporate fluids from the
surface of the chip.
[0108] Furthermore, multipoint calibrations may be performed if a
complete washing apparatus is attached to the chip surface, where
deionized water and increasingly concentrated salt solutions are
deposited, or flowed, onto the chip surface, and then a puff of air
is used to clear the array. At boxes 1912 and 1914, the sample
fluid is deposited onto the micro electrode array and the
calibrated osmolarity measurement is completed in the methods that
have been previously discussed.
[0109] Biomarker Normalization
[0110] In most patients who suffer from dry eye syndrome (DES),
ocular allergy, general or ocular infections, blepharitis,
diabetes, or other diseases in which DNA or other molecular
biomarkers are present in tears, there is a clear clinical need for
the ability to analyze nanoliter amounts of tears collected from
the lower tear lake.
[0111] Nanoliter tear samples are necessary to minimize the time of
residence of a collection device within the tear lake, which lowers
the chance of inducing reflex tearing, a situation in which
hypo-osmolar (less concentrated, very watery) tears are flushed
onto the ocular surface, thereby reducing the available biomarker
concentrations and introducing diagnostic variability within the
clinical routine. As the amount of reflex tearing is
disease-specific and patient-specific, the amount of dilution
varies with stimulation. Historically, tear collection protocols
suggest collecting relatively large volumes of tears, typically
several microliters of tears, in order to collect a sufficient
sample volume to conduct standard in vitro diagnostic tests. These
biomarker assays often take upwards of thirty minutes of continual
tear sampling to attain such high volumes. Older patients, and
especially those with DES, often present less than 200 nL of
available tears in the tear lake for sampling at a given time.
Thus, tear collection for standard in vitro tests is uncomfortable
and moderately invasive.
[0112] Hypo-osmolar tears can result from a variety of conditions.
An overabundance of non-lubricating tears can occur in certain dry
eye subtypes; known as epiphora, these patients may have occluded
nasolacrimal ducts which increase tear residence time within the
tear lake.
[0113] Patients with DES are also known to have a dysfunction of
the tear film that can result in a hyper-osmolar tear. Whether
through aqueous deficiency or meibornian gland disease, the steady
state equilibrium concentration of tears is significantly increased
in DES patients. Some DES patients are known to have steady state
tear lake concentrations approximately 30%-50% higher than
age-matched normals (healthy subjects). Measured osmolarities of
400 mOsm/L in the tear lake of severe DES patients have been
frequently reported. Hyper-osmolar tears are also observed in
contact lens wearers. Regardless of contact lens material or the
type of lens worn, contact lenses disrupt the preocular tear film
and shift the homeostasis of tears towards a hyper-osmolar
state.
[0114] Post-LASIK patients and DES patients may also have varying
levels of innervation and/or nerve function, which affect the
ability to produce reflex tearing. In vitro diagnostics performed
on these types of patients may therefore report quite different
concentrations of biomarkers depending on the state of the patient
and the manner in which tears are collected. There is a clear need
for in vitro diagnostic methods that can eliminate the variability
introduced by tear sampling and from hypo-osmolar and hyper-osmolar
tear film concentrations.
[0115] Recently, a new class of microfluidic technologies have
greatly reduced the volume requirements for in vitro diagnostics,
wherein submicroliter samples can be used to test for biomarkers
within tears. Because the tears offer an ideal, largely acellular
biological matrix from which to perform various in vitro
diagnostics, collecting tears may now be of interest to many
doctors and medical professionals who are less familiar with
working near the ocular surface, and who may unknowingly cause
undue reflex tearing during tear collection. The undue reflex
tearing from such sampling can cause inaccurate diagnostic results.
This problem reinforces the need for techniques of measuring
biomarker concentrations in tears that are independent of
sampling.
[0116] In accordance with the present invention, biomarker
normalization is performed against a measured osmolarity in order
to remove the impact of tear sampling and patient-specific tear
homeostasis from the interpretation of biomarker concentration in
tears. The normalization provides an Adjusted Tear Biomarker
Level.
[0117] Traditional measurement of tear biomarkers such as
immunoglobulins (IgE, IgA, IgG, IgM), glucose, insulin levels,
lactoferrin, tear lysozyme, cytokines, hormones, hormone
metabolites, infectious disease phenotypes, nucleic acids,
proteins, or lipid fractions, are carried out without the
simultaneous analysis of tear osmolarity. Traditional means of
measuring tear osmolarity are incompatible with tear biomarker
analysis. In accordance with the present invention, a receiving
substrate, sample region, and energy transduction mechanism within
a nanofluidic channel provide for the first time, the possibility
of measuring tear osmolarity on the same undiluted tear sample as
the biomarker. The combination of an integrated tear collection
interface and transducer provides leverage against evaporation
following sampling.
[0118] The calculation of an Adjusted Tear Biomarker Level is as
follows. Normal tear osmolarity is generally accepted to be near
300 mOsm/L (with ranges from 280-316 mOsm/L). The Adjusted Tear
Biomarker Level can be obtained from the following equation:
Adjusted Tear Biomarker Level=(300 mOsm/L*Measured Tear Biomarker
Level)/(Measured Tear Osmolarity Level)
The defined value of 300 mOsm/L can be substituted for any of the
appropriate range of tear osmolarity levels. In another embodiment,
the basal level of tear osmolarity can be measured for a specific
patient at the beginning of a study, at pretreatment, at an early
age, or prior to surgery in order to establish a personalized
baseline level of tear homeostasis. Following the passage of time,
a pharmaceutical administration, or surgery, the personalized
baseline level can be substituted for the defined 300 mOsm/L.
[0119] FIG. 20 is a flowchart that illustrates processing in
accordance with the normalization technique described herein.
Initially, at the box numbered 2002, an aliquot volume (such as a
nanoliter tear volume) of sample fluid is collected to a sample
region of a sample chip. Next, at box 2004, an osmolarity output
signal is received from the sample region that indicates energy
properties of the sample fluid, wherein the osmolarity output
signal is correlated with osmolarity of the sample fluid. Next, at
box 2006, a biomarker output signal is received from the sample
region that indicates chemical properties of the sample fluid,
wherein the biomarker output signal is correlated with biomarker
concentration of the sample fluid. Next, at box 2008, the
osmolarity output signal is processed to produce an osmolarity
value for the sample fluid and the biomarker output signal is
processed to produce a biomarker concentration value for the sample
fluid. The processing can be performed simultaneously or serially.
Lastly, at box 2010, the Adjusted Biomarker Level is determined,
which provides normalization of biomarker concentration values. As
noted above, the adjusted level provides a normalization of
biomarker concentration values and can correct for patient-specific
tear homeostasis and clinician induced tear sampling variance in
connection with obtaining the sample fluid.
[0120] The operations depicted in FIG. 20 can be performed by any
of the system embodiments illustrated in the drawings (FIGS. 1-11)
with a processor configured to perform the normalization operations
as described herein.
[0121] If desired, open loop adjustment is also possible, where the
300 mOsm/L constant is unused, as in the following equation:
Open Loop Adjusted Tear Biomarker Level=Measured Tear Biomarker
Level/(Measured Tear Osmolarity Level)
[0122] An advantage of using a standard or personal baseline
osmolarity value to normalize against is that the Adjusted Tear
Biomarker Level is expressed in units identical to the Measured
Tear Biomarker Level. Open loop adjustment would result in a
Biomarker Level/mOsms/L, which could be a more difficult parameter
for clinicians to interpret, especially if the analyte of interest
is commonly known to have a range in unnormalized units.
[0123] Similar Adjusted Tear Biomarker Levels can be performed
using linear, logarithmic, exponential, or through the use of
calibration curve adjustments. For example, a linear adjusted Tear
Biomarker Level could take on the form given by:
Linear Adjusted Tear Biomarker
Level=B.sub.adj=B.sub.m*(1+(Alpha*(Osm.sub.m-300 mOsms/L)))
where B.sub.adj is the Linear Adjusted Tear Biomarker Level,
B.sub.m is the measured biomarker level, Alpha is the linear
correction factor, and Osm.sub.m is the measured osmolarity. Both
the Alpha and the 300 mOsms/L point can be altered to fit the
specific biomarker curve.
[0124] IgE, for example, is suggested to be found on the order of
50-60 of ng/mL range in unsensitized individuals, and 100-300 ng/mL
in patients with vernal, seasonal, or perennial conjunctivitis (see
publications by Nomura, "Tear IgE Concentrations in Allergic
Conjunctivitis" in Eye, Vol. 12 (Part 2), 1998 at 296-98; and
Allansmith, "Tissue, Tear, and Serum IgE Concentrations in Vernal
Conjunctivitis" in Am. J. of Ophthalmology, Vol 81, No. 4, 1976, at
506-11). From Nomura: [0125] Tear IgE concentrations showed
significant increases in the vernal keratoconjunctivitis
(322.2+/-45.7 ng/ml), seasonal allergic conjunctivitis
(194.7+/-21.7 ng/ml) and perennial allergic conjunctivitis
(134.8+/-23.1 ng/ml) groups when compared with controls (52.1+/-9.7
ng/ml, p<0.01). No significant difference was found between
epidemic keratoconjunctivitis (97.2+/-11.7 ng/ml) and bacterial
conjunctivitis (92.6+/-13.8 ng/ml) groups and controls (p=0.1).
[0126] For DES patients with an elevated osmolarity of 400 mOsm/L,
unnormalized determination of the tear IgE levels may easily lead
to incorrect interpretation. A more severe bacterial conjunctivitis
could easily be mistaken for a relatively mild perennial allergic
conjunctivitis based on unnormalized IgE. Similarly, if a normal
patient with seasonal allergic conjunctivitis was overstimulated
during tear collection and produced hypo-osmolar reflex tears,
their tear IgE levels could easily drop beneath perennial allergic
conjunctivitis indications. Normalizing by measured tear osmolarity
prevents this type of misdiagnosis.
[0127] In one embodiment, a plurality of electrodes contained
within the sample region of the receiving substrate can be
functionalized with distinct energy transduction mechanisms; one
set of electrodes would contain an osmolarity transducer (e.g., a
non-polarizing metal electrode for impedance analysis of osmolarity
such as gold, platinum, and the like, and a conductive polymer such
as polypyrrole, polyacetylene, polyaniline, and the like) with
another set of electrodes configured as an electrochemical
transducer for a specific biomarker (e.g., a sandwich or
competitive assay comprising a bare metal or conductive polymer
coated electrode with corresponding surface chemistry to bind
antibody, avibody, aptamer, or other receptor for a the biomarker
ligand). In this embodiment, the osmolarity is multiplexed in
space. An example of this embodiment is shown in FIG. 21, which is
a plan view of the receiving substrate 2100 showing the sample
region 2102. A detail view of the electrodes 2104 in the sample
region 2102 is provided in FIG. 22. The illustrated electrodes 2104
indicate a group of electrodes demarcated within the sample region
as group "A" with the biomarker function and gold osmolarity
electrodes demarcated within the sample region as group "B" for the
osmolarity function. A capillary 2106 receives the sample fluid and
distributes the fluid along its length for interaction with the
electrodes A and B.
[0128] Upon depositing an aliquot volume of sample fluid on the
sample region of a substrate (through capillary action, aspiration,
or similar techniques), energy imparted into the sample fluid is
transduced by the sample region to produce an output signal that
indicates the energy properties of the sample fluid that are
correlated with the osmolarity of the sample fluid. Simultaneously
or in parallel operations, potentiometric, amperometric, pulse
voltammetry, cyclic voltammetry, broadband frequency response,
impedance, or other electrochemical methods are used to transduce
output signals from the electrochemically modified electrodes to
indicate chemical properties of the sample fluid that are
correlated with the concentration of biomarkers in tears. Thus, the
osmolarity and biomarker output signals are generated at the same
time but from different sets of electrodes. Subsequently, an
Adjusted Tear Biomarker Level is calculated to compensate for the
possibility of patient hyperosmolarity or dilution introduced by
tear sampling. That is, the Adjusted Tear Biomarker Level can
compensate and correct for patient-specific tear homeostasis and
for clinician-induced tear sampling variance in connection with
obtaining the sample fluid.
[0129] In other embodiments where osmolarity is multiplexed in
space, optical indicators, such as a plurality of nano-scale
spheres having a luminescence correlated to osmolarity of the
sample fluid are deposited on a subset of the sample region. Other
optical transduction mechanisms can include iontophoretic
fluorescent nanoscale spheres, or metal films amenable to surface
plasmon resonance. In parallel, subsets of the sample region are
configured to produce output signals that indicate chemical
properties of the sample fluid that are correlated with the
concentration of a biomarker in tear. Sample region subsets can
include luminescence, fluorescent, chemiluminescent, resonant
energy transfer, optoentropic, surface enhanced Raman,
colorimetric, surface plasmon resonant, plasmonic, or other optical
indicators commonly used for biomarker transduction. Following
illumination by an optical energy source that imparts optical
energy into the sample fluid, the optical energy can be transduced
by the sample region to produce an optical output signal that
indicates the energy and chemical properties of the sample fluid
that are correlated with the osmolarity and tear biomarker
concentration, respectively. An optical detector then receives the
optical output signal from the sample region, and a processing
device processes the output signal to produce an estimate of sample
fluid osmolarity and biomarker concentration. Subsequently, an
Adjusted Tear Biomarker Level is calculated to compensate for the
possibility of patient hyperosmolarity or dilution introduced by
tear sampling.
[0130] In yet another embodiment, electrical, optical, or thermal
(e.g., freezing point depression) methods of osmolarity
determination within the receiving substrate can be independently
combined with electrical or optical methods of tear biomarker
concentration detection. For example, conductive osmolarity
determinations can be combined with optical transducers for tear
biomarker analysis. Spatial multiplexing supports multiple
biomarkers in such a format.
[0131] In spatial multiplexing embodiments, the measurement of tear
osmolarity can either be performed at the same time as the
biomarker assays, or serially by modulating the input energy
type.
[0132] For example, if both osmolarity and tear biomarker analysis
are spatially multiplexed via optical methods, then tear osmolarity
can be determined by surface plasmon resonance (i.e., the angle
atop a metal film) and the tear biomarker can be analyzed by
fluorescence.
[0133] In another embodiment, electrodes covered with a chromogenic
competitive assay system can be interrogated for conductivity in
order to determine osmolarity, followed by absorbance of light in
order to quantify the concentration of tear biomarker.
[0134] If fluorescent nanoscale spheres are used as an osmolarity
marker and chemiluminescent reporter antibodies are used to
transduce the tear biomarker concentration, then the first input
would comprise an appropriate excitation light, and the second
energy input would comprise pumping a known concentration of
luminescent substrate and fuel across the sample region (e.g.,
luminol and hydrogen peroxide).
[0135] In another embodiment, a "molecular ruler" could be used to
transduce the osmolarity, for example, a plasmonic pair of
nanoscale metal spheres attached to DNA could indicate the bulk
sample fluid osmolarity by the optical detection of absorbance
change around 520 nm. In parallel, if fluorescently labeled
secondary antibodies are used to label the analyte of interest, the
fluorescent response from excitation light would be read following
the absorbance of the molecular ruler within the same fluid.
[0136] Other combinations of electrical, optical, and thermal
transduction can be combined to achieve requisite levels of
sensitivity, specificity, and multiplexing while minimizing the
need for washing or external interfacing to the sample region.
[0137] These methods are generally amenable to spatial multiplexing
in a discrete sense, where subsets of the sample region are
orthogonal within the surface plane. Such methods are also amenable
to vertical spatial multiplexing, where, for example, the biomarker
transducer is built atop the osmolarity transducer, as in a
fluorescent assay built atop a conductive polymer.
[0138] In another embodiment, a plurality of electrodes are
configured for electrochemical transduction of the biomarker of
interest, and the osmolarity is multiplexed in time. In this
embodiment, all the electrodes are functionalized with the same
surface chemistry for the biomarker assay. Because there is a
diffusion time associated with the ligand binding of the tear
biomarker, osmolarity can be determined immediately after
introduction into the sample region, prior to the electrodes being
substantially affected by the presence of analyte. In one
embodiment, electrochemical assays where the Debye layer is
modulated by the tear biomarker assay and is detected by a change
is capacitance of the system, the baseline reading can be
correlated to tear osmolarity, and the dynamic change in
capacitance over time can indicate the levels of tear biomarker.
Thus, the osmolarity and biomarker output signals are produced from
the same electrodes but are separated in time, the osmolarity
output occurring substantially immediately upon introduction of the
sample fluid to the sample region and the biomarker output
occurring following the requisite diffusion time for the sample
region.
[0139] Other embodiments allow for the osmolarity to be determined
at a different frequency spectrum than the biomarker assay. For
example, the osmolarity can be determined by a 10-100 kHz impedance
spectra, and the tear biomarker concentration analyzed by a DC or
low frequency amperometric or voltammetric steady state
measurement. Alternatively, the osmolarity can be determined by a
10-100 kHz impedance spectra, and the tear biomarker concentration
analyzed by a 100 kHz-GHz excited nanostructure spectra, or THz
adsorption spectra. Other combinations of pulsed, or sinusoidal
electrochemical measurements, including the addition of a small
sinusoidal signal atop a square wave input, can be used for such
analyses.
[0140] Other aspects in accordance with the invention can include
analysis of tear osmolarity and tear biomarkers to be analyzed in
parallel nanofluidic chambers, and then normalized against each
other.
[0141] Still other aspects of the invention include for the
implementation where two separate tear samples are taken and
analyzed in serial. Serial analyses of tear biomarker and tear
osmolarity would give an indirect estimate the impact of sampling.
It is likely that sequential analysis, if performed properly, would
give a better indication of the tear homeostasis than unnormalized
biomarker analysis alone.
[0142] While certain embodiments have been described above, it will
be understood that the embodiments described are by way of example
only. Accordingly, the inventions should not be limited based on
the described embodiments. Rather, the scope of the inventions
described herein should only be limited in light of the claims that
follow when taken in conjunction with the above description and
accompanying drawings.
* * * * *