U.S. patent application number 14/558058 was filed with the patent office on 2015-06-04 for integrated type microfluidic electrochemical biosensor system and method for rapid biochemical analysis.
The applicant listed for this patent is SHANGHAI INSTITUTE OF APPLIED PHYSICS, CHINESE ACADEMY OF SCIENCES. Invention is credited to Chunhai FAN, Qing HUANG, Fan YANG, Xiaolei ZUO.
Application Number | 20150153300 14/558058 |
Document ID | / |
Family ID | 50167132 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150153300 |
Kind Code |
A1 |
FAN; Chunhai ; et
al. |
June 4, 2015 |
INTEGRATED TYPE MICROFLUIDIC ELECTROCHEMICAL BIOSENSOR SYSTEM AND
METHOD FOR RAPID BIOCHEMICAL ANALYSIS
Abstract
The present invention provides an integrated type microfluidic
electrochemical biosensor system for rapid biochemical analysis and
the usage of the system. The system comprising: a continuous
feeding unit for sequentially conveying lead eluent, sample
solution, sample eluent, signal probe solution, signal probe eluent
and electrochemical detection buffer solution; a microfluidic chip
consists of one or more micro-channel network, the microfluidic
chip covers the electrode array to form a channel system, capture
probes which have interaction with the said sample solution fixed
on the surface of the electrode array, said channel system is
connected with the continuous feed unit; and a power system for
providing power to said continuous feeding unit. The invention
innovatively combine three technologies of planar electrode arrays,
microfluidic chip technology and continuous feeding unit together,
and the integrated type microfluidic electrochemical biosensing
system which is small in size and low in cost and has a wide
application prospect is provided.
Inventors: |
FAN; Chunhai; (Shanghai,
CN) ; YANG; Fan; (Shanghai, CN) ; ZUO;
Xiaolei; (Shanghai, CN) ; HUANG; Qing;
(Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SHANGHAI INSTITUTE OF APPLIED PHYSICS, CHINESE ACADEMY OF
SCIENCES |
Shanghai |
|
CN |
|
|
Family ID: |
50167132 |
Appl. No.: |
14/558058 |
Filed: |
December 2, 2014 |
Current U.S.
Class: |
205/775 ;
204/403.01 |
Current CPC
Class: |
B29C 66/112 20130101;
B01L 2400/049 20130101; B29L 2031/756 20130101; B01L 2200/12
20130101; B01L 2300/0645 20130101; B29C 66/71 20130101; B01L
2300/087 20130101; B01L 2300/0636 20130101; B29C 66/53461 20130101;
B29C 66/114 20130101; B29C 66/919 20130101; B29C 66/949 20130101;
B29C 65/02 20130101; B01L 2200/0689 20130101; B01L 2200/0673
20130101; G01N 27/44791 20130101; G01N 35/08 20130101; B01L
2300/0887 20130101; B01L 3/502784 20130101; B29C 66/028 20130101;
B01L 2300/0825 20130101; B29C 66/1122 20130101; G01N 27/44713
20130101; B01L 2300/0816 20130101; B29C 66/71 20130101; B29K
2083/00 20130101 |
International
Class: |
G01N 27/28 20060101
G01N027/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 2, 2013 |
CN |
201310638228.9 |
Claims
1. An integrated type type microfluidic electrochemical biosensor
system for rapid biochemical analysis, characterized by comprising:
a continuous feeding unit for sequentially conveying lead eluent,
sample solution, sample eluent, signal probe solution, signal probe
eluent and electrochemical detection buffer solution; a
microfluidic chip consists of one or more micro-channel network,
the microfluidic chip covers the electrode array to form a channel
system, capture probes which have interaction with the said sample
solution fixed on the surface of the electrode array, said channel
system is connected with the continuous feed unit; and a power
system for providing power to said continuous feeding unit.
2. The microfluidic electrochemical biosensor system according to
claim 1, wherein The microfluidic chip and electrode array formed
no-leakage reversible or irreversible channel system by plasma
cleaning and heat bonding process.
3. The microfluidic electrochemical biosensor system according to
claim 2, wherein the condition of the heat bonding process is
heated above 37.degree. C. for more than 30 minutes.
4. The microfluidic electrochemical biosensor system according to
claim 1, wherein the electrode array is a carbon electrode array or
gold electrode array prepared by the screen printing technique, or
an electrode array produced by directly depositing nano metal
particles on the surface of the carbon electrode electrochemical
deposition of the electrode array or the nano metal, or a planar
electrode array produced by photolithography technique.
5. The microfluidic electrochemical biosensor system according to
claim 1, wherein the capture probes fixed on the surface of the
electrode array include antibodies, antigens, nucleic acids or
nucleic acid aptamers.
6. The microfluidic electrochemical biosensor system according to
claim 1, wherein the continuous feeding unit is consists of small
tube which have through channel, and said lead eluent, sample
solution, sample eluent, signal probe solution, signal probe eluent
and electrochemical detection buffer solution spaced by the air
bubble continuously went through the through channel into the
micro-channel network of said microfluidic chip.
7. The microfluidic electrochemical biosensor system according to
claim 1, wherein The power system is consists of the injection pump
or injector connecting the downstream of the microfluidic chip and
provide vacuum negative pressure as the fluid driving force to
achieve the automatic transfer of solutions with different
functions of within the continuous feeding unit.
8. An usage of integrated type microfluidic electrochemical
biosensor system for rapid biochemical analysis, which is
characterized by comprising: providing an integrated type
microfluidic electrochemical biosensor system according to claim 1;
connecting the power system to the downstream of the microfluidic
chip, sequentially conveying lead eluent, sample solution, sample
eluent, signal probe solution, signal probe eluent and
electrochemical detection buffer solution into said microfluidic
chip's micro-channel network by said continuous feeding unit and
interacting with the capture probe fixed on the surface of the
electrode array, achieving continuous capture and cleaning,
generating a detectable electrochemical signal quickly, reading out
electrochemical signal of the electrode array surface modified by
different capture probes once by electrochemical workstation.
9. The usage according to claim 8, wherein the lead eluent, the
sample solution, the sample eluate, the signal probe solution,
signal probe elution, and the electrochemical detection buffer
solution sequentially spaced by air bubbles with length of 0.5 cm
or more to prevent cross-contamination.
10. The usage according to claim 9, wherein the power system is
consists of the injection pump or injector connecting the
downstream of the microfluidic chip, and said injection pump or
injector connect to the outlet end of said microfluidic chip by a
period of small rubber tube which have strong deformability to form
a vacuum negative pressure system.
Description
TECHNICAL FIELD
[0001] The present invention relates to a microfluidic
electrochemical biosensor systems, and more particularly relates to
an integrated type microfluidic electrochemical biosensor system
and method for rapid biochemical analysis.
BACKGROUND
[0002] The electrochemical biosensor is an apparatus using the
specific which is particular to biochemical reactions to
selectively identify a particular substance to be detected and
converting the biochemical reaction into electrical signal output.
Electrochemical detection method itself has some unique advantages,
including: rapid detection, high sensitivity, high selectivity,
simple instrumentation, easy miniaturization, integration, low
power consumption, and suitable for field testing. However, the
traditional three-electrode system, such as carbon electrodes and
gold electrodes and so on, not only throughput is low, the cost is
also higher. It is difficult to meet the high-throughput and
low-cost testing requirements at the present stage. Recently, the
rapid development of printing technology and lithography greatly
contributed to the development of high-throughput and disposable
electrodes, but the long time sample incubating and time consuming,
tedious cleaning steps limit the further application of such
electrochemical biosensor. Microfluidic chip technology can
effectively solve the above problems.
[0003] Microfluidic chip analysis is based on analytical chemistry
and biochemistry and relied on MEMS processing technology,
characterized by micro-channel network structure. The parts of the
collection of the sample, pretreatment, separation, reaction,
detection and the like are integrated type within the scope of a
few square centimeters to complete the processing and testing of
samples fast and efficiently. Because the micro-channel depth,
width are in the micron level, it can be effective to limit the
target to be detected and the capture probes which have fixed
sensor interface in the scope of micrometer scales for interaction,
greatly increases the ability to identify molecules.
[0004] Rapid molecular recognition of the electrode surface is the
foundation of efficient scene electrochemical biosensing. Soh, et
al. (Swensen, J. S.; Xiao, Y.; Ferguson, B. S.; Lubin, A. A.; Lai,
R. Y.; Heeger, A. J.; Plaxco, K. W.; Soh, H. T., Continuous,
Real-Time Monitoring of Cocaine in Undiluted Blood Serum via a
Microfluidic, Electrochemical Aptamer-Based Sensor. JACSj
2009,131,4262-4266) prepared flat gold electrode by lithography,
constructed microfluidic E-DNA sensor. However, the cost of
preparation of the electrode lithography is too high, the mass
production is difficult. In addition, Rusling et al.
(Chikkaveeraiah, B. V.; Mani, V.; Patel, V.; Gutkind, J. S.;
Rusling, J. F., Microfluidic electrochemical immunoarray for
ultrasensitive detection of two cancer biomarker proteins in serum.
Biosens. Bioelectron, 2011, 26, 4477-4483) using a printing
electrode array and a microchannel structure to fix machine screws
and plexiglass clamps to seal the sensor chip reversibly. Although
a super-sensitive markers detection is achieved, each time the
sample tests need to re-assemble and disassemble sensors and insert
counter electrode and reference electrode along the microchannel
repeatedly. The workload is heavy and tedious. Although there are
reports of a single-electrode microfluidic electrochemical sensor
drived by capillary force, but the force can not complete delivery
of various solutions and is difficult to carry out multivariate
detection (Lillehoj, P. B.; Wei, F.; Ho, C.-M., A self-pumping
lab-on-a-chip for rapid detection of botulinum toxin. Lab Chip,
2010, 10, 2265-2270). How to achieve efficient molecular
recognition and complete delivery of a variety of solution rapidly
remains a serious challenge. Recently, Sia, et al. (Chin, C. D.;
Laksanasopin, T.; Cheung, Y. K.; Steinmiller, D.; Linder, V.;
Parsa, H.; Wang, J.; Moore, H.; Rouse, R.; Umviligihozo, G.;
Karita, E.; Mwambarangwe, L.; Braunstein, S. L.; van de Wijgert,
J.; Sahabo, R.; Justman, J. E.; El-Sadr, W.; Sia, S. K.,
Microfluidics-based diagnostics of infectious diseases in the
developing world. Nat. Med., 2011,17,1015-1019) completed rapid
detection of infectious diseases by introducing gas interval
solution zone and a microfluidic chip, but the method is mainly
carried out by introducing enhanced signal to analyze the disease
qualitatively, the clinical and field testing need for more
quantitative analysis of disease markers.
SUMMARY OF THE INVENTION
[0005] The object of the present invention is to provide an
integrated type microfluidic electrochemical biosensor system and
method for rapid biochemical analysis, so as to solve defect that
the prior art fails to quickly complete delivery of various
solutions, microfluidic chip's micro channel network layer and the
electrode array layer is fixed by a auxiliary equipment, and the
defect that can not achieve quantitative analysis of disease
markers, and electrochemical biosensor system of the prior art is
restricted to planar electrode array prepared by lithographic
techniques, resulting in the problem of high cost.
[0006] To solve the above problems, the present invention employs
the following technical solutions.
[0007] The present invention provides an integrated type
biochemical microfluidic electrochemical biosensor system for rapid
biochemical analysis, comprising: a continuous feeding unit for
sequentially conveying lead eluent, sample solution, sample eluent,
signal probe solution, signal probe eluent and electrochemical
detection buffer solution; a microfluidic chip consists of one or
more micro-channel network, the microfluidic chip covers the
electrode array to form a channel system, capture probes which have
interaction with the said sample solution fixed on the surface of
the electrode array, said channel system is connected with the
continuous feed unit; and a power system for providing power to
said continuous feeding unit.
[0008] Under the action of the power system, said continuous
feeding unit sequentially conveying lead eluent, sample solution,
sample eluent, signal probe solution, signal probe eluent and
electrochemical detection buffer solution into said microfluidic
chip's micro-channel network and interacting with the capture probe
fixed on the surface of the electrode array, generating an signal
detectable by electrochemical device, reading out electrochemical
signal of the electrode array surface modified by different capture
probes once.
[0009] The microfluidic chip and electrode array formed no-leakage
reversible or irreversible channel system by plasma cleaning and
heat bonding process.
[0010] The condition of the heat bonding process is heated above
37.degree. C. for more than 30 minutes.
[0011] The electrode array is a carbon electrode array or gold
electrode array prepared by the screen printing technique, or an
electrode array produced by directly depositing nano metal
particles on the surface of the carbon electrode electrochemical
deposition of the electrode array or the nano metal, or a planar
electrode array produced by photolithography technique.
[0012] The capture probes fixed on the surface of the electrode
array include antibodies, antigens, nucleic acids or nucleic acid
aptamers.
[0013] The continuous feeding unit is consists of small tube which
have through channel, and said lead eluent, sample solution, sample
eluent, signal probe solution, signal probe eluent and
electrochemical detection buffer solution spaced by the air bubble
continuously went through the through channel into the
micro-channel network of said microfluidic chip.
[0014] The power system is consists of the injection pump or
injector connecting the downstream of the microfluidic chip and
provide vacuum negative pressure as the fluid driving force to
achieve the automatic transfer of solutions with different
functions of within the continuous feeding unit.
[0015] The invention also provides an usage of an integrated type
microfluidic electrochemical biosensor system for rapid biochemical
analysis, comprising: providing a microfluidic electrochemical
biosensor system as described above; connecting the power system to
the downstream of the microfluidic chip, sequentially conveying
lead eluent, sample solution, sample eluent, signal probe solution,
signal probe eluent and electrochemical detection buffer solution
into said microfluidic chip's micro-channel network by said
continuous feeding unit and interacting with the capture probe
fixed on the surface of the electrode array, achieving continuous
capture and cleaning, generating a detectable electrochemical
signal quickly, reading out electrochemical signal of the electrode
array surface modified by different capture probes once by
electrochemical workstation.
[0016] The lead eluent, the sample solution, the sample eluate, the
signal probe solution, signal probe elution, and the
electrochemical detection buffer solution sequentially spaced by
air bubbles with length of 0.5 cm or more to prevent
cross-contamination.
[0017] The power system is consists of the injection pump or
injector connecting the downstream of the microfluidic chip, and
said injection pump or injector connect to the outlet end of said
microfluidic chip by a period of small rubber tube which have
strong deformability to form a vacuum negative pressure system.
[0018] The invention innovatively combine three technologies of
planar electrode arrays, microfluidic chip technology and
continuous feeding unit together to form a micro-scale sensing
method for simultaneous detecting multiple disease markers.
Electrochemical analysis and microfluidic chip is actually belong
to two different study fields. Electrochemical analysis field
emphasis on the interface design and regulation of electrode, not
much on manipulation, continuous feeding or continuous cleaning and
other aspects of the sample. The field of microfluidics stressed
controllable manipulation of fluid and micro-channel chip design to
match or meet the appropriate means of detection. Although
integrated type electrochemical detection microfluidic chip has
long been reported, but more concentrated in a glass or silicon
substrate photolithography planar microelectrodes. The main reason
for this is the photolithography planar microelectrodes can match
the micro-channel chip, and easy bonding. But the cost of using
such photolithography to prepare electrode is high, it is difficult
to mass production. In addition, these integrated type
electrochemical detection microfluidic chips mainly cumbersome
feeding and cleaning by capillary force or power provided by the
micro-pump. And the present application is applicable not only to
the electrode array has been successfully commercialized as a
macroscopic printed electrode of plastic substrate, but also
extends to the plane macro-electrode prepared by the
photolithography technique. We successfully combine the printed
electrodes which have infinite market with a new microfluidic
technologies and integrated type disposable continuous feeding
unit, with significant innovation and application prospects.
[0019] The invention is advantageous over the prior art has the
following effects:
[0020] I) can convey a variety of solution once quickly, to flow
through micro-scale sensor interface continuously, in order to
improve the efficiency of molecular recognition, operate easily,
fast, greatly simplify the procedure of electrochemical
biosensors;
[0021] 2) the micro-channel network of the microfluidic chip and
the electrode array layer achieved no-leakage seal by processing,
do not need any auxiliary equipment which is used to fix, reducing
the difficulty of preparing the device, increasing the
reproducibility and stability of preparation.
[0022] 3) can achieved quantitative detection with different
concentrations of a variety of different target, less reagent
consumption, rapid analysis;
[0023] 4) an integrated type microfluidic electrochemical
biosensing system which is small in size and low in cost and has a
wide application prospect is provided.
BRIEF DESCRIPTION OF FIGURES
[0024] FIG. 1 is a perspective diagram illustrating the structure
of a microfluidic electrochemical biosensor system according to a
preferred embodiment of the present invention;
[0025] FIG. 2 is a sectional view of a microfluidic electrochemical
biosensor system which is shown in FIG. 1;
[0026] FIG. 3 is a schematic diagram of the process based on the
formation of a single three-electrode system microfluidic
biosensor;
[0027] FIG. 4 is a schematic diagram of the process based on the
formation of four three-electrode system microfluidic
biosensors;
[0028] FIG. 5 is a schematic diagram of the surface process and
bonding procedure of the microfluidic electrochemical biosensor
based on printed electrode array and the polydimethylsiloxane
micro-channel;
[0029] FIGS. 6A and 6B are comparison chart for bonding single
three-electrode system and the micro-channel network of the
microfluidic chip to detect 500 ng/mLPSA current;
[0030] FIG. 7 is a result chart for split vaccine of human
influenza virus H1N1 is detected in the single three-electrode
microfluidic electrochemical biosensor detection system;
[0031] FIG. 8 is a result chart for human prostate cancer marker
PSA of different concentration is detected in the microfluidic
electrochemical biosensor system shown in FIG. 1;
[0032] FIG. 9 is a result chart for human liver cancer marker AFP
of different concentration is detected in the microfluidic
electrochemical biosensor system shown in FIG. 1.
EMBODIMENT
[0033] The following Examples with reference to specific
embodiments, the present invention is further described below. It
should be understood, the following examples only illustrate the
present invention and not for limiting the scope of the
invention.
[0034] As shown in FIG. 1-FIG. 2, a microfluidic electrochemical
biosensor system according to a preferred embodiment of the present
invention comprising: a continuous feeding unit 1 for the convey
solution of different function, a microfluidic chip 2 consisting of
four micro-channel network 21, an electrode array 3 covered by said
microfluidic chip 2, a power system 4 is connected with the
downstream of the microfluidic chip 2.
[0035] The continuous feeding unit 1 is formed of a transparent
plastic small tube 11 to provide a through passage for the solution
of different function sequentially to go through the passage into
the micro-channel network 21 of the network 2, solution of
different function is spaced by air bubble 13 to form different
functional solution zones 12. Preferably, the length of the air
bubbles 13 is to be maintained at more than 0.5 cm to prevent the
former solution and later solution from mixing to form a
cross-contamination in the feeding and loading procedure due to
extrusion of air bubble 13 or discontinuous dispersion of solution
caused by rapid changes in pressure.
[0036] Referring to FIG. 3-FIG. 4, preferably, the microfluidic
chip 2 is made of four serpentine micro-channel network 21, the
electrode array 3 is composed of an four three-electrode system
printed electrode. The four electrode are working electrode 31,
counter electrode 32, reference electrode 33, and a printed
electrode silver wire 34 (see FIG. 1). FIG. 5 shows the surface
process and bonding procedure of the microfluidic chip 2 and the
electrode array 3. First, the microfluidic chip 2 network 21
comprising the micro-channel network 2 (made of
polydimethylsiloxane (PDMS)) and the electrode array 3 optionally
protected by PDMS frame are processed by plasma. After that, PDMS
frame which have surface antibody activity and protect the
electrode array is removed so that PDMS channel layer and the
electrode array layer are aligned and thermal bonding. Since the
surface of the chip and the surface of the electrode array are
produced by plasma treatment, a large number of oxygen-containing
functional groups are formed. Oxygen-containing groups in the
interface cross-linking react to produce irreversible chip bonding,
so that the microfluidic chip and the electrode array can form a no
leakage reversible or irreversible system without machine screws
and the upper and lower splints and any outside force. Preferably,
the condition of the heat bonding process is preferably heated
above 37.degree. C. for 30 min.
[0037] The power system 4, as a power source of continuous feeding
unit 1, is is connected with the downstream of the microfluidic
chip 2. The preferred embodiment use disposable syringe 41. When
using the syringe by pulling the push handle 41 to a certain height
and then fixed by wooden strips or metal rods to form vacuum
negative pressure in the whole flow channel. The negative pressure
used as the driving force of the fluid so as to realize an
automatic transfer of various functional solution zones 12 in the
continuous feeding unit 1. In order to ensure the smooth formation
of vacuum negative pressure, the syringe 41 is connected with the
outlet end of microfluidic chip 2 through a period of a piston
rubber small tube 42 which has strong deformation and matching
diameter. The piston rubber small tube 42 is designed with
adjustable valve 43 to control the opening and closing of the whole
flow channel. Syringe 41 and the rubber small tube 42, also the
rubber small tube 42 and the outlet end of the microfluidic chip 2
is further connected through a suitable small pipe.
EXAMPLE 1
[0038] This embodiment use a microfluidic electrochemical biosensor
(FIG. 3) formed before and after the bonding of a single
three-electrode unit and the micro-channel network to detect human
prostate cancer marker PSA. Specific steps are as follows: A 20
.mu.L TMB solution (3,3',5,5'-tetramethylbenzidine hydrochloride)
were added to the surface of three-electrode (Normal) and
micro-channels covered by PDMS (Channel), then the electrode
connect to the electrochemical workstation to do the cyclic
voltammetry test and obtain the experiment results in FIG. 6A.
[0039] We use 1 mL syringe to continuous manual extract horseradish
peroxidase-conjugated avidin (avidin-HRP), water, buffer solution
(0.01M phosphate, 0.14M NaCl, 2.7 mM KCl, pH7.2), the
biotin-labeled PSA (biotin-PSA) and 500 ng/mL PSA solution zone,
there are air bubbles which have 0.5-1 cm length between the
solution zone interval, where the volume of each solution zone is
1-20 .mu.L; connect the prepared microfluidic chip, continuous
feeding unit, the power system into an entirety, injection pump
extraction flow rate was adjusted to 1-20 .mu.L/min, when the
solution area in the continuous feeding unit to be seen began to
flow to the microfluidic chip, pausing the extraction to quickly
adjust the flow rate to 2-5 .mu.L/min. During the continuous
flowing process of the solution zone, the PSA-Ab (monoclonal
antibodies) fixed on the electrode interface successively binding
500 ng/mL PSA, 10-20 .mu.g/mL biotin-labeled second antibody
biotin-PSA in the sample zone to form the sandwich structure and
couple with the avidin-HRP in the signal probe solution zone. After
use the buffer solution and water to wash off unbound probe
complex, directly dropping 10 .mu.L TMB solution in the inlet
without power injection, the electrode is connected with an
electrochemical workstation to do the amperometric detection, HRP
enzyme catalyze H.sub.2O.sub.2 in the TMB solution to amplify
electrochemical signal circularly to obtain the experimental
results of FIG. 6B.
EXAMPLE 2
[0040] This embodiment also use a microfluidic electrochemical
biosensor (FIG. 3) formed before and after the bonding of a single
three-electrode unit and the micro-channel network to detect split
vaccine of human influenza viruses H1N1. Steps are as follows: use
1 mL syringe to continuous manual extract H1N1-HRP, water, buffer
solution (0.01M phosphate, 0.14M NaCl, 2.7 mM KCl, pH7.2), and H1N1
split vaccine solution zone of a certain concentration, there are
air bubbles which have 0.5-1 cm length between the solution zone
interval, where the volume of each solution zone is 1-20 .mu.L;
connect the prepared microfluidic chip, continuous feeding unit,
the power system into an entirety, injection pump extraction flow
rate was adjusted to 1-20 .mu.L/min, when the solution area in the
continuous feeding unit to be seen began to flow to the
microfluidic chip, pausing the extraction to quickly adjust the
flow rate to 2-5 .mu.L/min. During the continuous flowing process
of the solution zone, the H1N1-77 (H1N1 antibodies) fixed on the
electrode interface successively binding 0-500 ng/mL H1N1, 10
.mu.g/mL HRP-labeled second antibody HRP-H1N1 (probe solution) in
the sample zone to form the sandwich structure. After use the
buffer solution and water to wash off unbound probe complex,
directly dropping 10 .mu.L TMB solution in the inlet without power
injection, the electrode is connected with an electrochemical
workstation to do the amperometric detection, HRP enzyme catalyze
H.sub.2O.sub.2 in the TMB solution to amplify electrochemical
signal circularly to obtain the experimental results of FIG. 7.
EXAMPLE 3
[0041] This embodiment use a microfluidic electrochemical biosensor
(FIG. 4) consists of the bonding of four three-electrode units and
the micro-channel network to detect human prostate cancer marker
PSA and human liver cancer marker AFP simultaneously. Steps are as
follows: use 1 mL syringe to continuous manual extract avidin-HRP,
water, buffer solution (0.01M phosphate, 0.14M NaCl, 2.7 mM KCl,
pH7.2), mixture of biotin-PSA and biotin-AFP, and PSA and AFP
antigen mixture solution zone of a certain concentration, there are
air bubbles which have 0.5-1 cm length between the solution zone
interval, where the volume of each solution zone is 1-20 .mu.L;
connect the prepared microfluidic chip, continuous feeding unit,
the power system into an entirety, injection pump extraction flow
rate was adjusted to 1-20 .mu.L/min, when the solution area in the
continuous feeding unit to be seen began to flow to the
microfluidic chip, pausing the extraction to quickly adjust the
flow rate to 2-5 .mu.L/min. During the continuous flowing process
of the solution zone, the PSA-Ab and AFP-Ab (monoclonal antibodies)
fixed on the electrode interface successively binding 0-100 ng/mL
PSA and 0-500 ng/mL AFP, 10-20 .mu.g/mL biotin-labeled second
antibody biotin-PSA and 12.5-25 .mu.g/mL biotin-labeled second
antibody biotin-AFP in the sample zone to form the sandwich
structure and couple with the avidin-HRP in the signal probe
solution zone. After use the buffer solution and water to wash off
unbound probe complex, directly dropping 20 .mu.L TMB solution in
the inlet without power injection, the electrode is connected with
an electrochemical workstation to do the amperometric detection,
HRP enzyme catalyze H.sub.2O.sub.2 in the TMB solution to amplify
electrochemical signal circularly to obtain the experimental
results of FIG. 8 and FIG. 9.
[0042] The above description is only the preferred embodiment of
the present invention, not intended to limit the scope of the
present invention. Various changes may be made to the
above-described embodiments of the present invention. All of the
simple, equivalent change and modification according based on the
claims of present application and specification content will fall
into the scope of protection required by the claims of patent. The
contents which are not detail in the present invention are all
conventional technical contents.
* * * * *