U.S. patent application number 11/475356 was filed with the patent office on 2010-09-23 for method of making biological components for devices by forced environmental adaptation.
Invention is credited to Jeffrey T. LaBelle, Vincent B. Pizziconi.
Application Number | 20100240020 11/475356 |
Document ID | / |
Family ID | 31978675 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240020 |
Kind Code |
A1 |
LaBelle; Jeffrey T. ; et
al. |
September 23, 2010 |
Method of making biological components for devices by forced
environmental adaptation
Abstract
An improved method for the design and development of high
performance biologically-derived components for use in hybrid
devices. The biologically-derived component is used in hybrid
constructs that may be nanostructures, given the small size of the
biological parts. Force adaptation is used to bring an organism
from which the biologically-derived component is developed to
provide such a component meeting a desired measure of performance.
In one specific embodiment, chlorosomes of Chloroflexus aurantiacus
(C. aurantiacus) enhance performance of a silicon photovoltaic
cell. C. aurantiacus, strain J-10-f1, has the A.T.C.C. designation
number 29366, having been deposited in July, 1976. Its chlorosomes
are harvested and positioned in light communicating relation to a
photoactive semiconductor.
Inventors: |
LaBelle; Jeffrey T.; (Tempe,
AZ) ; Pizziconi; Vincent B.; (Phoenix, AZ) |
Correspondence
Address: |
GALLAGHER & KENNEDY, P. A.
2575 E. CAMELBACK RD. #1100
PHOENIX
AZ
85016
US
|
Family ID: |
31978675 |
Appl. No.: |
11/475356 |
Filed: |
June 26, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10658541 |
Sep 8, 2003 |
7067293 |
|
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11475356 |
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60408775 |
Sep 7, 2002 |
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Current U.S.
Class: |
435/3 ;
435/252.1 |
Current CPC
Class: |
B01L 3/5085 20130101;
B01L 2300/168 20130101; G01N 21/31 20130101 |
Class at
Publication: |
435/3 ;
435/252.1 |
International
Class: |
C12Q 3/00 20060101
C12Q003/00; C12N 1/20 20060101 C12N001/20 |
Goverment Interests
STATEMENT OF GOVERNMENT FUNDING
[0003] Financial assistance for this project was provided by the
U.S. Government through the National Science Foundation Under Grant
Numbers 9602258 and 9986614 and the United States Government may
own certain rights to this invention.
Claims
1. A method of making biologically-derived components for hybrid
devices having biological and nonbiological components comprising:
(a) for the desired performance of the biologically-derived
components deriving a measure of performance, (b) producing
adaptable organisms from which the biologically-derived components
are to be developed, (c) controlling a plurality of environmental
factors under which the organisms are produced: (i) subjecting the
organisms being produced to several alternate values of several
controlled environmental factors, (d) monitoring the measure of
performance of the biologically-derived components developed from
the organisms produced in step (b), (e) repeating steps (b), (c)
and (d) until a desired measure of performance of the
biologically-derived components has been achieved.
2. The method according to claim 1, wherein the measure of
performance is a figure of merit.
3. The method according to claim 1, wherein the measure of
performance is a transfer function.
4. The method according to claim 1, wherein step (b) comprises
providing a multiple input, multiple output environmental chamber
and growing the components therein.
5. The method according to claim 4, wherein step (d) comprises
applying a design of experiments analysis to the environmental
factors and the measure of performance.
6. The method according to claim 1, wherein step (c) comprises
controlling a plurality of environmental factors chosen from the
group consisting of temperature, illumination, media and duration
in and during which specimens of the organisms are developed from
which the biologically-derived components are derived.
7. The method according to claim 1, wherein the organisms of step
(b) are bacteria.
8. The method according to claim 7, further comprising gathering,
as the biologically-derived components, selected parts of the
bacteria active in a mode of interest.
9. The method according to claim 8, wherein the selected parts of
the bacteria are chlorosomes.
10. The method according to claim 9, wherein the bacteria are
Chloroflexus aurantiacus (C. aurantiacus), and the chlorosomes are
RC.sup.- chlorosomes.
11. The method according to claim 1, wherein step (b) comprises
growing Chloroflexus aurantiacus (C. aurantiacus).
12. The method according to claim 11, further comprising gathering
the chlorosomes RC.sup.- from the C. aurantiacus for use in the
biologically-derived component.
13. A component, comprising one or more light antenna structures
biologically-derived from photosynthetic bacteria, for a hybrid
device, the component made by the method of any one of claims 1-12.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application off of U.S.
application Ser. No. 10/658,541 in the name of Jeffrey T. LaBelle
and Vincent B. Pizziconi, filed Sep. 8, 2003, entitled
"Nanoengineered Biophotonic Hybrid Device," which claims priority
from provisional U.S. patent application Ser. No. 60/408,775, of
the same inventors, filed Sep. 7, 2002 entitled "Method &
Apparatus for Synthesis, Processing, Design & Manufacturing of
High Performance, Scalable & Adaptive, Robust
Energy-Interactive Materials, Devices & Systems." Priority from
each of the foregoing two applications is hereby claimed. Each of
the foregoing two applications is incorporated herein by
reference.
[0002] This application is related to three concurrently filed
applications, each in the name of the present applicants and all
sharing priority from the above-identified provisional and parent
U.S. utility application Ser. No. 10/658,541. The three
concurrently filed applications are entitled "Nanoengineered
Biophotonic Hybrid Device," "Device With Biological Component And
Method Of Making To Achieve A Desired Figure of Merit," and "Device
With Biological Component And Method of Making To Achieve A Desired
Transfer Function."
FIELD OF THE INVENTION
[0004] This invention relates to hybrid biological and electronic
photosensitive devices and more particularly to nanoscale hybrid
devices of this kind and their method of manufacture.
BACKGROUND
[0005] Recently, attempts to marry biology and engineering to
create various biohybrid constructs have been steadily increasing.
A limited number of novel biohybrid sensor applications have
already been reported, and in some cases commercialized, that
incorporate "smart" molecular-scale biological components. These
have attracted considerable interest from both the biomedical and
biotechnology communities worldwide. However, little has been done
to date in developing integrated nanodevices and systems such as
microanalytical systems incorporating novel, engineered
nanobioconstructs and their analogues for use in integrated
nanodevices and systems such as bio-optical hybrid sensors capable
of very sensitive and selective nanoscale detection due to enhanced
performance characteristics as determined by a prescribed biohybrid
Figure of Merit (FoM). Potential applications include microsystem
applications requiring low-level light detection capability (e.g.
micro total analytical systems (.mu.TAS) for immunoassay, genomics
and proteomics), such as "point-of-care" diagnostic medicine,
biotechnology, space bioengineering, and countermeasures to
biowarfare for defense.
[0006] In general, the current state of art for engineering design
as taught by Koen (Koen, 1987) and many others (Otto and Wood,
2001), has not led to the achievement of device components,
stand-alone devices, nor engineered systems that function or
otherwise perform at a prescribed FoM and oftentimes typically
perform at levels significantly below optimal FoM levels and
theoretically achievable maximum FoM limits.
[0007] The well known area of thermoelectric device design
exemplifies the present ability of engineering design heuristics to
achieve a desirable thermoelectric FoM (i.e., ZT) that
significantly exceeds current ZT device values of .about.1 although
a ZT value of 4 is theoretically possible (Rowe, D. M., 1995). The
present inability by those skilled in the art to achieve desired
material and device FoM's is essentially true for virtually all
engineering device design applications spanning diverse
disciplinary fields and broad industry product segments.
[0008] In recent years, less effective and predictive empirical
approaches have been used to devise novel hybrid devices that
incorporate naturally-derived, or mimetics thereof, biological
materials and constructs that have resulted in enhanced device
performance relative to their non-hybrid engineered counterpart. To
date, however, the engineering method does not teach how to design,
select, modify or otherwise alter smart, nanoscale
energy-interactive materials (e.g., molecular-scale biophotonic
components) derived from natural or biomimetic analog constructs, a
priori, in spite of their intrinsically superior and potentially
adaptable structural and performance characteristics. These
materials are referred to herein as "biologically-derived," meaning
in whole or in part grown as an organism or as part of an organism,
in whole or in part extracted from an organism (whether altered or
unaltered, adapted or unadapted) or biomimetic analogues of the
foregoing. The engineering method referred to above does not show
those skilled in this art how such nanoscale materials can be
further embodied or employed as components, or as stand-alone
devices, that are capable of producing robust and scalable
energy-interactive biohybrid devices and systems, a priori, to
function at a desired FoM not yet achievable by conventional
engineering means.
[0009] Photoactive semiconductors such as Si photovoltaic cells (as
one example of a large scale device) have long been known. They
have been employed in various devices and applications for years.
Their varying responsivity to certain light wavelengths throughout
the visible spectrum has been observed as well. On the biological
side, thermophilic photosynthetic bacteria such as Chloroflexus
aurantiacus (C. aurantiacus) and other species have been studied
and reported upon. The photosensitive "antenna" cells,
"chlorosomes," of these organisms have been studied and reported
upon, as well. Perhaps as a result of inconsistency of results with
photosynthetic bacteria, these organisms and their chlorosomes have
not been incorporated into useable devices. There has been no
successful synthesis of photosensitive semiconductor materials with
the chlorosomes of photosynthetic bacteria reported. A need exists
for improvement of the performance of photoactive devices
throughout the light spectrum, and for techniques for using the
good photosensitivity of photosynthetic bacteria in photoactive
devices. More fundamentally, there is a need to identify
inconsistencies in the photosensitivity (or other photonic or
electroactivity) of biological specimens and to apply a method or
methods to ameliorate or eliminate such inconsistencies.
[0010] As one means of gathering knowledge about a system, Design
of Experiment (DOE) analysis is a widely used statistical modeling
approach, reported in detail elsewhere (Montgomery, 1991). A unique
advantage of DOE, particularly as applied to complex adaptive
systems, is its ability to elucidate, not only the effect of the
controlling variables, but also their complex interactions. Use of
DOE analysis with biological or hybrid biological/nonbiological
devices and systems has not been encountered. In particular use of
the powerful DOE approach in connection with forced adaptation in
biological systems (such as bacteria) to move the systems toward a
more consistent (i.e. dependable) performance is not known. Figure
of Merit (FoM) is another concept often used in engineering (among
other fields such as economics, chemistry, astronomy, etc.). FoM is
a measure of a device's performance. It is used in many contexts.
However FoM as a design-driving measure, particularly with respect
to adaptive biological organisms-based systems, devices and
components is considered to be a radical departure from other uses
of this concept. Further, as applied to biological organisms, parts
thereof or systems made up of such organisms, a means to control
multiple environmental variables is needed if the DOE approach is
to be applied.
[0011] The transfer function of a device, circuit or system is
another engineering concept that is well understood. However, that
concept has not ordinarily been applied to biological systems, if
at all. A need exists to apply engineering concepts like DOE, FoM
and the transfer function to the analysis, evaluation and design of
biological, bioengineered and hybrid systems, components and
devices.
SUMMARY
[0012] Broadly, the present invention encompasses equipment and
methods for the synthesis, processing, design and manufacturing of
high performance, scalable, adaptive and robust energy-interactive
hybrid materials, devices and systems combining biological and
nonbiological technologies. Specifically, an exemplary embodiment
of the invention adapts powerful engineering concepts to the
engineering of biological components that are to be used in
manufactured devices and systems, including hybrid devices and
systems.
[0013] FIG. 1 exemplifies a novel method that will guide those
skilled in the art to achieve the design and development of high
performance hybrid materials and devices. As illustrated, several
key steps are depicted in FIG. 1 that show one skilled in the art
how to achieve desired and even optimal hybrid device designs that
utilize smart, nanoscale constructs acquired, harvested or
otherwise derived directly from complex living organisms. A
preferred embodiment is the use of a multiple input-multiple output
apparatus, such as a multiple input-multiple output environmental
chamber (i.e., MIMO/EC), and applicable computational algorithms to
extract useful and exploitable hybrid device design heuristics. Use
of this method will result in a desired and prescribed Figure of
Merit in spite of the use of previously unknown or poorly defined
or characterized nanoscale biological constructs and their
function. In applied form, the novel engineering design method
described herein will provide a means to identify or otherwise
exploit intractable, or very difficult to identify, useful
engineering specifications. A preferred embodiment of this
invention is shown in FIG. 2, an illustration of a novel method and
apparatus for the design and development of high performance hybrid
materials and devices.
[0014] One application of the proposed invention is the enhancement
of well-known photoactive semiconductor devices, such as Si
photovoltaic cells using nanoscale biophotonic constructs that are
either acquired, harvested or otherwise manipulated in their
natural or adapted state using the method and apparati described
herein to achieve desired FoM performance characteristics. Although
commercially available Si photovoltaic cells have been employed in
various devices and applications for years, their FoMs are
typically low despite detailed knowledge of their structure and
function and the ability to prescribe device performance
specifications from use of selected light wavelengths throughout
the visible spectrum, as well as, related device specifications
associated with the engineering transfer function.
[0015] The transfer function of a component, device, or system is a
useful engineering concept, directly related to the FoM, that is
well known and understood by those skilled in the art. However, the
use of a transfer function and related FoM concepts have not been
generally applied and prescribed to biological constructs intended
for use in the design of biohybrid devices and systems, if at all.
Thus, an unmet and nonobvious need still exists to use well known
engineering heuristics such as, the design of experiments (i.e.,
DOE), FoM and the transfer function for the analysis, design, and
evaluation of bioengineered hybrid components, devices and
systems.
[0016] To demonstrate the novelty and utility in the use of the
hybrid device design heuristic to achieve high performance hybrid
materials and devices (FIG. 2), the invention described herein
improves the device performance (i.e., the FoM) of a stand-alone,
commercial silicon photovoltaic device (Si PV) using a nanoscale
bioderived construct with generally unknown engineering
specifications. However, the methods and apparati taught herein
generally apply to the design and exploitation any smart nanoscale
or integrative nanoscale material, construct, or system, or mimics
thereof, that is amenable to the FoM enhancement of a hybrid device
or system.
[0017] A typical FoM of a Si PV device is generally less than 1 and
typically only .about.0.28-0.32. Although a number of potentially
useful hybrid design approaches can be employed to improve the SiPV
FoM using the invention taught herein, the use of a nanoscale
biophotonic construct having desired complementary energy transfer
properties constitutes a potentially viable hybrid design approach.
One such nanoscale biophotonic construct having potentially useful
and exploitable engineering specifications to enhance the FoM of a
photonic device, such as a SiPV device, is the nanoscale
pigment-protein supramolecular construct known as a light antenna
structure that function as energy funnels in thermophilic
photosynthetic bacteria such as Chloroflexus aurantiacus (C.
aurantiacus) and other photosynthetic species. These highly quantum
efficient photosensitive constructs (also known as "chlorosomes")
are known to perform significant photonic energy shifts (red
shift). In the case of the chlorosome associated with the C.
aurantiacus, an input photonic energy at a wavelength of
.about.460-480 nm is typically shifted to .about.800-820 nm with
very little energy loss. Hence, as described, the chlorosomes and
the photovoltaic cell are each energy-interactive or energy
converting, the chlorosomes converting electromagnetic energy
(light) at a first range of wavelengths to electromagnetic energy
(light) at a second range of wavelengths, and the cell converting
electromagnetic energy (light) to electrical energy. Combined, they
provide an example of the high performance, scalable, adaptive, and
robust energy-interactive hybrid devices referred to above.
[0018] Typically SiPV devices are more sensitive to higher photonic
wavelengths and generally most sensitive to the near infrared
region (i.e. 800-900 nm) of the electromagnetic spectra. Thus, in
principle, the use of biologically-derived light antenna
structures, as well as mimics or analogs thereof, could potentially
enhance the FoM of a Si PV device if exploitable engineering
specification(s), such as the transfer function or its associated
FoM, could be identified, acquired, developed and subsequently
employed successfully in a SiPV engineered hybrid device or system
that meets a prescribed and verifiable FoM that validates the
desired performance of the hybrid biophotonic device. However, the
achievement of desired FoMs using hybrid device and system
approaches is not obvious to those skilled in the art of device
design and development and empirical combinations of smart
materials or components used in the design and manufacture hybrid
device can oftentimes lead to device and system performances (i.e.,
FoM) inferior to nonhybrid device and system counterparts.
[0019] A preferred embodiment of the invention makes use of
well-known design algorithms, such as the Design of Experiment
(DOE), among many others known and appreciated by those skilled in
the art. DOE analysis is a widely used statistical modeling design
tool reported in detail elsewhere (Montgomery, 1991). A unique
advantage of DOE, particularly as applied to complex adaptive
systems, is its ability to elucidate, not only the effect of the
controlling or independent variables, but also their oftentimes
complex interactions.
[0020] The use of DOE analysis in combination with a novel MIMO/EC
apparatus can be used to identify, acquire or otherwise produce
useful and exploitable engineering hybrid device and system
specifications from complex biological constructs in their isolated
or natural state or environment, or mimics thereof. In particular,
the combined use of DOE with the MIMO/EC apparatus can provide a
novel and powerful design heuristic to achieve desired engineering
specifications of nanoscale-based constructs via their
identification and/or modification from complex, adaptive systems,
such as, viable organisms.
[0021] The use of the DOE-MIMO/EC apparatus in this embodiment is
most useful when it may be desirable to modify one or more
properties of a complex, adaptive construct through the `forced
adaptation` of a modifiable biological component of a viable,
complex systems (such as bacteria). This produces the desired
modification of a potentially useful property or characteristic of
e.g. a nanoscale-based component that is useful to achieve a
desired performance level (i.e. FoM) of a device or system in which
that is not otherwise achievable by its nonhybrid.
[0022] In one embodiment of the invention by varying the
environmental conditions under which a biological component of, for
example, a hybrid device, is grown, a transfer function for that
component can be altered. Using the MIMO/EC of this invention, a
biological component may be force adapted in such a manner as to
affect a modification of a transfer function that governs its
outputs under given inputs. The desired transfer function can thus
be engineered into a biological component, within bounds.
[0023] In one exemplary embodiment of the invention, the methods
and equipment of the invention are used to engineer an exemplary
hybrid photoactive component. That component combines a hitherto
acceptable photoactive semiconductor device with a biological
mechanism that has extreme high photoactive performance to achieve
performance unprecedented in devices of the type. This hybrid
device, itself an exemplary and preferred embodiment of one aspect
of this invention, uses a constituent of a photosynthetic bacterium
to enhance the response of a semiconductor photoactive device
across the intended spectrum of its use.
[0024] With the methods and equipment of this invention,
chlorosomes of the thermophilic green photosynthetic bacterium
Chloroflexus aurantiacus (C. aurantiacus) are successfully coupled
to a photoactive semiconductor device to derive enhanced
performance across the relevant spectrum.
[0025] Here, using design of experiment (DOE) methodology, adaptive
biological units, such as cells, are force-adapted to achieve
consistent performance in the characteristics of interest. In this,
a multiple input-multiple output environment chamber (the
above-mentioned MIMO/EC) affords the ability to force adapt the
bacteria from which these chlorosomess are gathered.
[0026] This embodiment is focused on exploiting biosystems at the
nanoscale for their utility as functional `device` components in a
proposed biohybrid microdevice. More specifically, a design
feasibility study was implemented to evaluate the efficacy of a
naturally occurring nanoscale biophotonic, light adaptive `antenna`
structure (the chlorosome), isolated from C. aurantiacus. The
overall objective was to assess its utility as functional device
component that would enhance the spectral performance
characteristics of well-characterized photonic devices, i.e.,
solid-state photovoltaic.
[0027] The chlorosomes are nanoscale, optical functional units
(100.times.30.times.10 nm). They can transfer photonic energy at
high quantum efficiencies (69-92%) and ultra-fast rates
(picoseconds). They were fabricated into programmed arrays on solid
substrates and fully characterized. These biological assemblies
were subsequently integrated with the well-characterized
photodetectors and evaluated for their potential to selectively
enhance performance the spectral regions where the photodetectors
are inherently insensitive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 is a conceptual block diagram illustrating elements
in the design and development of a device, in particular a hybrid
device of biological and nonbiological content;
[0029] FIG. 2 is a conceptual flow chart of the design process for
designing a hybrid device using a multiple input, multiple output
environmental chamber and employing a figure of merit to gauge the
performance of a biological component;
[0030] FIG. 3. is an image of C. aurantiacus by a scanning electron
microscope;
[0031] FIG. 4 is a cartoon schematic rendering of chlorosomes of C.
aurantiacus in place in a cytoplasmic membrane;
[0032] FIG. 5 is a diagrammatic (cartoon) illustration of a
chlorosome of the bacterium C. aurantiacus with its four major
subunits (the chlorosome designated herein the RC.sup.+
chlorosome);
[0033] FIG. 6 is a diagrammatic (cartoon) illustration of the
chlorosome of the bacterium C. aurantiacus of FIG. 5, but with two
of its four subunits, the B808/866 protein light harvesting
apparati and a reaction center removed (the chlorosome thus
modified designated herein the RC.sup.- chlorosome);
[0034] FIG. 7 is a diagrammatic (cartoon) illustration of the
chlorosome of FIG. 6 with parts broken away for clarity showing
contained rod-like structures of Bchl c;
[0035] FIG. 8 is a functional block diagram in the form of a flow
chart of optical interactions of the components of the chlorosome
shown in FIG. 5;
[0036] FIG. 9 is a plot of absorbent spectra data for a C.
aurantiacus chlorosome;
[0037] FIG. 9A is an exemplary normalized absorbance spectra plot
of the RC.sup.- chlorosome;
[0038] FIG. 10 is a diagrammatic block diagram in the form of a
flow chart indicating the optical interaction of the parts of the
chlorosome of FIG. 6;
[0039] FIG. 11 is a diagrammatic illustration, partly in section,
of a hybrid photovoltaic device in accordance with the
invention;
[0040] FIG. 12 is an enlarged fragmentary cross-sectional view
along the line 12-12 of FIG. 11 and shows the chlorosomes like that
of FIG. 6 adherent to a transparent plate;
[0041] FIG. 13a is a normal percentage probability plot and FIG.
13b is the interaction plot between temperature and percent volume
for a design of experiments analysis where the output variable to
be studied was the ratio R1 of absorbance at 740 nm to absorbance
at 808 nm;
[0042] FIG. 14a is the normal percent probability plot and FIG. 14b
the interaction plot between temperature and percent volume media
to air for a design of experiments analysis where the output
variable studied is the ratio R2 of absorbance at 740 nm to
absorbance at 366 nm;
[0043] FIG. 15a is a plot of three replicates of a full spectra of
C. aurantiacus at one dilution and FIG. 15b plots full spectra of
absorbance of C. aurantiacus at multiple concentrations;
[0044] FIG. 16a is a plot of correlation between absorbance and
cell count at 650 nm wavelength for C. aurantiacus and FIG. 16b is
a plot of correlation between absorbance and cell count at 740 nm
wavelength;
[0045] FIG. 17 is a plot of correlation between absorbance and
number of RC.sup.- chlorosomes of C. aurantiacus at 650 nm
wavelength and a zoomed-in-plot of the first four data points in
that correlation showing close linearity between the two
variables;
[0046] FIG. 18 is a plot of absorbance and emissions spectra of
chlorosomes of C. aurantiacus;
[0047] FIG. 19 is a plot of percent enhancement of a SiPV for
percent coverage by chlorosomes of C. aurantiacus;
[0048] FIG. 20 is a functional block diagram that illustrates the
use of a figure of merit in the development of a biological hybrid
device with feedback from the Figure of Merit determination through
a DOE or the like development program and feedback from the device
performance;
[0049] FIG. 21 is a formula for the photonic figure of merit
devised for C. aurantiacus, a tabulation of the measures going into
that formula for seven specimens and a block diagram illustrating
the interaction of the major contributing factors to the figure of
merit;
[0050] FIG. 22 is a diagrammatic illustration of a multiple input,
multiple output environmental chamber having nine individual
compartments; and
[0051] FIG. 23 a perspective view of an environmental chamber like
that diagrammatically illustrated in FIG. 22
DETAILED DESCRIPTION
[0052] The bacteria, Chloroflexus aurantiacus (C. aurantiacus),
strain J-10-f1, has the American Type Culture Collection (ATCC)
designation number 29366, having been deposited in July, 1976. The
ATCC is located at 10801 University Boulevard, Manassas, Va.
20110-2209 U.S.A. The C. aurantiacus bacteria is a green,
nonsulfur, flexing/gliding, photosynthetic bacteria. It is
thermophilic and can be found in hot springs up to temperatures of
70.degree. C. in large mat-like layers. The layers, when
concentrated enough, have an orange coloration.
[0053] A freeze fracture image of C. aurantiacus by scanning
electron microscopy (SEM) was taken and is reproduced in FIG. 3. In
the image small ovals can be resolved. These are the cell's
chlorosomes. At this size scale reduction would require specialized
EM or other imaging techniques. Thus far, no high resolution
structural information has been successfully obtained on individual
chlorosome structures, and as such a cartoon schematic
representation of the chlorosomes 100 in situ is presented in FIG.
4. There, the chlorosomes 100 are depicted in place in a
cytoplasmic membrane 95. A proposed model of a single chlorosome
100 is shown enlarged in FIG. 5 in a 3-D cartoon. From the work of
Blankenship, et al., the chlorosome 100 is comprised of four major
sub-units: a Bchl c portion 101, a Bchl baseplate 102, B808/866
protein, supra molecular light harvesting complex or apparati 103,
and a reaction center (RC) 104.
[0054] A chlorosome 110 of the C. aurantiacus bacterium is depicted
in FIG. 6. It includes two major supra-molecular pigment-protein
subunits. These are the bacteriochlorophyll (Bchl) c 101, and the
supra-molecular baseplate complex 102. In its form shown in Fig.
the C. aurantiacus chlorosome 100 is here designated RC.sup.+
(meaning with its RC 104 and B808/866 light harvesting apparati 103
in place). As depicted in FIG. 6 at 110, stripped of its associated
reaction center and B808/866 supra-molecular complex 103, the
chlorosome of C. aurantiacus is designated RC.sup.- (meaning
without the RC 104 and B808/866 light harvesting apparati 103).
Each sub-unit of the chlorosome 100 illustrated in FIG. 5 is
composed of a large number of wavelength-specific light absorbing
and transducing molecules.
[0055] The first sub-unit involved in light transduction is a lipid
sack 101 containing bacteriochlorophyll (Bchl) c, which is
organized in units of approximately 10,000 molecules that form
rod-like structures 115 (FIG. 7). As represented in the flow chart
of FIG. 8 at 115, these molecules transduce photonic energy
associated with 740 to 750 nm light in approximately 16 ps with
very little loss. Photonic energy at 750 nm is then transduced at
117 by the membrane of the baseplate 102, which is comprised of
approximately 500 molecules of Bchl a, to 795 nm to 800/810 nm in
41-175 ps. The B808/866 complex 103 contains 10-20 Bchl a
molecules, which absorb at 119 at 808 and 866 nm and transfer at
883 nm in approximately 250 ps. Finally, the last stage is where,
at 121, a special pair of Bchl a molecules of the reaction center
(RC) 104, convert the light energy into chemical (photochemistry)
to emit photons.
[0056] FIG. 9 plots absorbance spectra data of isolated chlorosomes
of C. aurantiacus noting peaks of interest. There, an absorbance
peak at 740-750 nm attributable to the Bchl c rods 113 appears. A
peak at 795 nm associated with the Bchl a baseplate is shown. In
addition absorption of light in the blue region by the cartenoids
is evident and blue secondary absorbance peaks from the Bchl c and
a (designated as Soret peaks) occur. A peak attributable to the
monomeric form of Bchl c (like its Soret) has a different
absorbance wavelength peak than the oligomeric form that comprises
the rods 113 in the chlorosomes. Like the Bchl a baseplate peak,
the Bchl c oligomeric c peak is in the near infrared (NIR).
[0057] Isolated RC.sup.- chlorosomes in Tris buffer exhibit the
absorbance peaks (solid line) shown in the normalized absorbance
spectral plot of FIG. 9A. Immobilizing the RC.sup.- chlorosomes in
PVAC polymer, however, destroyed the chlorosomes as evidenced by
the dashed line normalized absorbance spectrum plotted in FIG. 9A.
This was true of other immobilization attempts with other
polymers.
[0058] Intact C. aurantiacus bacteria display a unique adaptive
ability to reversibly and enzymatically assemble and disassemble
the foregoing structures to protect the organism from photo-induced
damage. As is expected, the spectral peaks of FIG. 9 are highly
related to growth conditions of the whole cell C. aurantiacus
bacteria. These are also related to the isolation techniques that
result in purified chlorosomes. An abbreviated form of the
important basic mechanisms of energy transfer that occur between
the molecules of the RC.sup.- chlorosome are as depicted in FIG.
10.
[0059] The carotenoids have been shown to also transfer energy to
the Bchl c oligomeric rods as is true of the Soret band (a strong
absorbance of a chlorophyll in the blue region of light). However,
there are subtle differences in the Bchl c found in the
chlorosomes. The Bchl c found in C. aurantiacus chlorosomes are
self-assembled (from monomeric form) into oligomeric rods. This
results in a shift of the normal Soret (and Q.sub.y) band into a
redder form. The Bchl a found in the baseplate also has a Soret
region in its photonic (blue) spectra. Carotenoids can begin to
quench the structures and should be closely watched, as this would
cause the device of this invention to operate at lower
efficiencies.
[0060] In the exemplary embodiment of the invention that was
successfully made and tested, the RC- chlorosomes were suspended in
a liquid which was then applied to the hydrophobic surface of a
borosilicate glass plate 118 as shown in FIGS. 11 and 12. It is the
basis 102 of the chlorosomes 110 that adhere to the surface of the
plate 118.
[0061] As shown in FIG. 11 the plate 118 is supported just above
the surface of a glass slide or substrate 120. An epoxy seal 121 is
formed about the edges of the plate 118. On or closely spaced above
the plate 118 a commercially available silicon photovoltaic cell is
supported. Illumination of the chlorosomes and the photovoltaic
cell 125 by an LED 127 produces a voltage across the output of the
photovoltaic cell 125 as can be observed by a multimeter 129.
[0062] As shown in FIG. 18, when excited with 430 nm, 460 nm and
470 nm, which is exactly where the silicon photovoltaic cells is
less sensitive, the RC.sup.- chlorosome emits at about 810 nm where
the silicon photovoltaic cell is sensitive. There is, therefore, a
spectral enhancement by the addition of the biological component
that is similar to that shown generally in FIG. 18.
[0063] In an exemplary laboratory prototype of the device, the SiPV
was an Edmond Optics NT53-371 photovoltaic cell. Slide 120 was a
Fischer microwells slide, part number 12-568-20 and the plate 118
was a Fischer cover glass, part number 12-541A.
[0064] The microslide employed allowed for relatively
straightforward application of the chlorosomes. This particular
slide has two frosted rings on its surface, one of which is
indicated at 131 in FIG. 11. The frosted ring was just sufficiently
high above the surface of the slide 120 that a drop of the liquid
suspension containing the chlorosomes was retained. The cover glass
118 was rested on the ring 131 and when the suspending liquid had
evaporated leaving the chlorosomes adherent to the hydrophobic
borosilicate cover glass surface as shown, the epoxy seal 121 was
applied. The microwells slide was useful in another respect. Having
two of the frosted rings 131, it permitted for the side-by-side
construction as illustrated in FIG. 11 and a control. The control
could be an identical silicon photovoltaic cell illuminated through
the slide 120 and a further glass 118 but absent the chlorosomes,
or the control could be as illustrated in FIG. 11 but having the
RC.sup.+ chlorosomes entrapped.
[0065] In the arrangement of FIG. 11, the RC.sup.- chlorosomes and
the light receiving surface of the photovoltaic cells were no more
than a millimeter apart. As indicated in FIG. 11, the construction
of the off-the-shelf photovoltaic cell placed the light receiving
surface 133 of the silicon semiconductor in a metal housing or can
135 to be exposed through a glass closure 137.
[0066] Characteristics of the biological component of the hybrid
device of FIG. 11 are set forth in Table 1.
TABLE-US-00001 TABLE 1 Schematic Pictures of chlorosomes + Si PV
Chlorosome Characteristics Size: 100 .times. 30 .times. 10 nm
Approx Rh: 33 nm (calculated) 41 nm (DLS) Energy Transfer: Strokes
Shift: 470-800/810 nm .DELTA..lamda.: 320 nm QE: 69-92% QE Delay
Time: 50 ps - 1 ns Orientation Control: Yes Number of particles: 4
.times. 10.sup.7-8 .times. 10.sup.9 chlorosomes Number of
molecules: 4 .times. 10.sup.15-810.sup.17
Experimental
Materials and Methods
[0067] First, the biological component (the RC.sup.- chlorosomes),
as well as controls, had to be isolated or purchased. Next, several
types of characterization had to be performed (and developed in
some cases) so that the device fabrication could be accomplished.
These involved many steps (and iterations) until sufficient
materials were readily available (in the correct form) for use in
the hybrid device configuration.
[0068] C. aurantiacus cells were grown in `D` media, under 6000 lux
50.degree. C. in a one liter bottle (FIG. 3.2). The `D` mixture is
as follows (all chemicals from Sigma): A mixture of 50.0 ml of the
medium D stock is added to distilled water with 2.0 gm Difco Yeast
Extract, 1.0 gm Glycylgylcine (freebase) adjusting the pH to 8.2.
This mixture is then autoclaved for 0.5 hr at 450.degree. C. The
medium D stock is prepared by mixing 40.0 ml of Nitch's Solution to
80.0 ml of the FeCl.sub.3 solution in 3.5 l of distilled water with
the following traces: 8.0 gm Nitrilotriacetic acid, 4.8 gm of
CaSO.sub.4.2H.sub.2O, 8.0 gm MgSO.sub.4.7H.sub.2O, 0.64 gm NaCl,
8.24 gm KNO.sub.3, 55.12 gm NaNO.sub.3, and 8.88 gm
Na.sub.2HPO.sub.4. The Nitch's solution is made by placing 0.5 ml
concentrated H.sub.2SO.sub.4, 2.28 gm MnSO.sub.4.H.sub.2O, 0.5 gm
ZnSO.sub.4.7H.sub.2O, 0.50 gm H.sub.3BO.sub.3, 0.025 gm
CuSO.sub.4.7H.sub.2O, 0.025 gm Na.sub.2MoO.sub.4.2H.sub.2O, and
0.045 gm CoCl.sub.2.6H.sub.2O into 1 liter of distilled water. This
should be stored refrigerated. The FeCl.sub.3 solution is prepared
by adding 0.2905 gm of FeCl.sub.3 (or 0.4842 gm
FeCl.sub.3.6H.sub.2O) to 1 liter distilled water and should also be
refrigerated.
[0069] RC.sup.- chlorosome isolation (Gerola, 1986) starts with
cells concentrated (600 ml) by centrifugation at 3,600.times.g for
60 min. 2M NaSCN with 10 mM ascorbic acid in 10 mM Pi buffer (6.5
ml monobasic: 43.5 ml dibasic phosphate buffer per liter) was added
to the weighed pellet in 4 ml/gm amounts. Cells were homogenized
10.times. in a cell disruptor/homogenizer (Fisher Scientific).
Disruption of cells was performed by (one) pass in a 4.degree. C.
stored French Press (ThermoSpectronic) cell with 20,000 psi. DNAse
I (Sigma) was added and the solution was incubated for 30 min at
room temperature. The solution was passed through the cell two more
times.
[0070] Cell debris was removed by pelleting at 3,600.times.g for 1
hr. A continuous sucrose gradient was established by placing 2.0 ml
of a 40% sucrose in the NaSCN buffer in a tube and layering on 3.0
ml of a 10% sucrose solution. The tubes were placed, horizontally,
into a dark, 5.degree. C. storage until use (48 hrs later). The
addition of 1.2 ml chlorosome solution to the top and
ultracentifugation at 144,000.times.g for 18 hrs was started. Bands
were collected by removal of the top band (by color), then removal
by 1 ml at a time until the pellet was reached. The pellet was
collected by addition of 1 ml to the tube and slight sonication to
homogenize the pellet).
[0071] After isolation of the RC.sup.- chlorosomes from C.
aurantiacus whole cells, the chlorosomes were (at various dilutions
into Tris buffer) tested as in the above methods. This was
performed in order to assess quality control by comparing spectral
data (on absorbance) and relative output (emission).
[0072] Surface effects (of the substrates) were tested by contact
angle goniometry. This required a flat substrate (of sufficient
surface area) to be placed on a Rame-Hart NRL Contact Angle
Goniometer and test the contact angle with solution of known
surface energies. Solution droplet formation was done with a
syringe and about 100 .mu.l droplet. Images were taken with the
instruments CCD and using the RHI Imaging 2001 software to capture
the digital pictures. Analysis can be done with the software. As
well, as images were printed out and results were manually
verified.
[0073] Another technique utilized the evaporation procedure as well
as an aqueous method to allow incorporation of the chlorosomes onto
a glass surface. Both techniques start with taking 0.5 .mu.l of a
known concentration of chlorosomes and placing it onto a
borosilicate glass coverslip (Fisher Scientific). In the
evaporation method, evaporation, under vacuum, is performed
overnight and then the sample is sealed onto a fluorescent antibody
microslide (Fisher Scientific). In the physical adsorption method,
the slide is prepared in the aqueous phase and inverted during
sealing, thus allowing for ensuring a hydrated sample as well as
diffusion of the chlorosomes onto the surface of the hydrophobic
glass. Samples were also studied under laser scanning confocal
microscopy (instrument from LEICA) to investigate orientation and
function (stability) was observed with absorbance spectroscopy of
the sample afterwards.
[0074] The engineering photonic devices had to be characterized.
Using a modified NIST approach to calibrate the detectors, a system
to develop sensitivity curves (to wavelength) was established. Each
device was calibrated under similar conditions and intensities were
varied to demonstrate intensity changes (if present) in the device.
Finally, devices that were to be enhanced had to be selected. Known
nonlinearities in traditional devices, such as the silicon PV's (or
solar cells) were selected as optimal devices for enhancement.
[0075] These devices were stimulated by white light filtered with
interference filters to provide wavelength control from blue to
NIR. Intensity was adjusted and matched using a radiometer from
International Light (IL-1700). Changing the intensity from 0.01 to
70 lux caused a dramatic shift in the blue region by use of NDF's
on an optical table (Edmund Optical Division).
[0076] The counting and size information gathering was accomplished
by several high-resolution microscopy techniques. Transmission
Electron Microscopy (TEM) was performed by taking isolated
chlorosomes and evaporating a 0.5 .mu.l drop onto a bacitracin
treated formvar coated grid (300 mesh). Negative stains of urinal
acetate were used to enhance the images. Images were taken at the
Life Science EM Facility at 25,000.times. magnification. Images
were saved in jpeg format, inserted into MATLAB and data (size and
counts) were taken. Calculations were then scaled to predict how
many chlorosomes were in a 1 ml sample for each of three dilutions.
Absorbance spectroscopy of these dilutions was also performed to
correlate absorbance spectra to count for the given population
using a Beckman DU-65 photospectrometer (technique mentioned
later).
[0077] A final method was used to gather most of the counting data,
namely, Field Emission Scanning Electron Microscopy (FESEM) at the
Center for Solid State Electronics Research Center at Arizona State
University on a Hitachi 4700 FESEM. Again, a hemocytometer
technique was employed as an initial method that could be
correlated to the others. Another technique used computer aided
image processing to allow the chlorosomes surface to be assigned a
`1` or `white` pixel value and the background a `0` or `black`.
Accounting for surface area (number of pixels) per chlorosome,
histograms were made and counts were calculated via computer. The
final technique was a modified ASTM method in which the surface is
transversed from left to right, and top to bottom, counting
chlorosomes until 100 is reached. Then the number of pictures
required to reach .about.100 chlorosomes, the surface area of each
picture, etc are accounted for and a final # chlorosomes/ml is
calculated. Here, five concentrations (plus a distilled water
control) were imaged using all three techniques and counts were
correlated to ABS spectra as well to aid in future calculations or
determinations. The stubs were prepared by evaporating 100 .mu.l of
the dilution onto a hydrophobic borosilicate glass disk, attaching
the disk to a stub via tape and carbon coating the samples for a
period of 10 minutes. The chlorosomes were diluted with Tris buffer
at pH 8.0 and 10 mM NaCl, by addition of 0.788 gm Trizma HCl into
500 ml of DI water, under constant stirring. Meanwhile, add 0.605
gm of Trizma Base was added into 500 ml of DI water under constant
stirring. Both solutions were mixed together and 0.9 gm NaCl was
added while mixture was stirred thus making 1 liter of 10 mM Trizma
buffer, pH 8.0 with 20 mM NaCl.
[0078] Another imaging technique, namely Atomic Force Microscopy
(AFM) was performed by evaporating a 100 .mu.l sample of
chlorosomes (overnight in desiccant jar) onto a standard
borosilicate coverglass. A Digital Instruments' Nanoscope III
Multimode AFM was used in Tapping Mode (TMAFM) to image the
chlorosomes at various dilutions. Again, the dilutions' absorbance
spectra were taken prior to imaging. Prior to running the AFM
experiments, a known liquid volume (400 .mu.l) was taken from
solution containing RC.sup.- chlorosomes in DI water previously
characterized via absorbance spectra (ABS=0.01@740 nm) and was
evaporated onto a clean, optically clear glass disk with known
surface area (113.1 mm.sup.2). The disks were made hydrophobic to
enhance RC.sup.- attachment and orientation due to theoretical
studies performed by using a molecular modeling algorithm (Chou,
1977) that suggested that the baseplate region attached to the
reaction center may be hydrophobic in nature. Tapping mode AFM
experiments were conducted utilizing a small scan head (D head) to
scan 1 .mu.m.sup.2 surface areas on both the control disks (no
RC.sup.- chlorosomes deposited) and test disks (RC.sup.-
chlorosomes deposited).
[0079] Ranges of dilutions were made by serial dilution of the
stock chlorosome sample. Each dilution was placed into a standard
cuvette (using a blank of Tris buffer) and full (400-900 nm)
absorbance readings were gathered (via an RS232C port) onto
computer and analyzed and plotted in MATLAB. At this point,
selection of a non-pigment wavelength (650 nm in the case of the
chlorosomes) was made to use in correlating absorbance to the
previous counts made on each dilution and then plotted. This
wavelength was selected for its non-photosynthetic (non-optically
active) properties and consistent nature between different growth
conditions during the counting experiments. Hence, a calibration
curve was made between counting and absorbance for a series of
dilutions of chlorosomes.
[0080] The TEM images were placed into the Image Processing Toolbox
for MATLAB for sizing measurements and calculations processing. The
scalebar was measured (in the number of pixels across it to length)
and then correlated to the size of the bar so a conversion could be
made for length and width of chlorosomes. The command `ginput` was
used to grab the distal ends of the chlorosomes and utilizing the
Pythagorean Theorem: a.sup.2+b.sup.2=c.sup.2, measurements of
length and width were made by selecting random chlorosomes and
measuring 5 per image. 25 chlorosomes from each image were selected
and measured to ensure statistical distributions could be made.
[0081] Counts (per .mu.m.sup.2) at this point were also made and
calculations were made to correlate to a count per ml of each
dilution and then related to the corresponding absorbance
spectra.
[0082] In the AFM and FESEM studied, the images were taken and
saved in jpeg format for processing in MATLAB as was done in the
TEM images. Size was verified but in these techniques, counting was
the main objective. The same process of taking the counts in an
area and re-calculating what the count per ml was performed on many
dilutions to enable a more accurate count (and correlation to
absorbance data).
[0083] The same samples were sent to Protein Solutions Inc. DLS was
run on the samples. Later (after purchase of a DLS system) 20.0
.mu.l of each sample was injected via a gas-tight syringe into the
quartz cuvette and readings were taken at 2 Acq/sec. Data filtering
was performed to minimize dust events but capture the quickly
diffusing small particles. The Dynamics V6 software developed the
autocorrelation curves and produced the polydisperse plots of
R.sub.h versus % mass for each sample.
[0084] During the AFM studies, the hydrophobicity of the baseplate
was studied by utilizing differently treated coverglass in LSC.
Untreated coverglass remained very hydrophobic, with a critical
surface tension around 12 dynes/cm, a surface tension of 32
dynes/cm, and a contact angle (for DI water) of 41.degree.. A heat
treatment (450.degree. C. for 4 hours) allowed for the coverglass
to pass through the glass-transition (T.sub.g) temperature and
delivers the surface into a hydrophilic state (surface tension
close to 12 dynes/cm and a contact angle of .ltoreq.1.degree. for
DI water). The samples were placed onto the surfaces (in a laminar
flow hood to reduce contamination) and evaporated under vacuum over
night. Imaging was performed within 36 hours of evaporation so that
the chlorosomes would not swell (degrade). Images were taken and
stored as jpeg format files and processed in MATLAB as with the
other imaging techniques. Unusual formations or interactions at the
surface were also imaged in the pictures. Placement of the
concentrations required to make certain percent coverages into the
microwells were done with an incubation time necessary for physical
adsorption. The time was a predicted time based upon diffusion
coefficient of the chlorosomes (as measured by DLS) and the path
length. The final, assembled coverglass and microwells were sealed
with a two-part epoxy and allowed to cure overnight.
[0085] The first stability test for the isolated chlorosomes tested
storage under two conditions. A `fresh` sample was maintained for
use in 7.degree. C. freezer and a long-term (or later called
`frozen`) sample was placed in liquid nitrogen (LN.sub.2). Initial
degradation was noted in the samples and can be clearly seen (at
the monomeric 670 nm absorbance peak) in the absorbance spectra of
the `fresh` sample. Emission spectra were even gathered to see if a
decrease in emission occurred.
[0086] In intensity related photodegradation, concentrations were
matched between all samples (six different intensities were
investigated) by absorbance readings. Therefore, a series of
experiments were designed and run with chlorosomes, with and
without reaction centers, in solution, to see this effect. Samples
were diluted to 1:100 of the original stock into Tris buffer. 2 ml
each were separated out for 6 different light conditions. Light
intensity was varied by the use of filters, no filter, or no light
such that % Transmissions were 0% T, 14% T, 36% T, 53% T, 68% T,
and 100% T and measured (photometrically). The light source was a
standard 100-watt white light bulb. Degradation was recorded at
times when 5, 10, and 15% degradation was noted. Degradation was
quantified by noting a percent decrease in the 740 nm absorbance.
The samples were continuously illuminated and at specific time
intervals, absorbance readings were taken. Degradation of the 740
Bchl c Q.sub.y band was measured by (1) peak height from start to
finish and by (2) integration of the area under the Q.sub.y band.
Times were marked when 5, 10, and 15% degradation of the peak were
attained. A control sample (buffer) was also held under the same
illumination and used as the blank in the photospectrometer.
[0087] Next, in intensity related to concentration photodegradation
experiments, various concentrations of chlorosomes (in 2 ml) were
degraded by a similar white light (at fixed intensity). From these
sets of experiments minimum photostress terms were calculated to
determine a 0% degradation intensity (and time). Again, the 740 nm
peak height provided a measure (by absorbance spectra) of
photodegradation over time.
[0088] Another mode of destruction of the photo-stability of the
chlorosomes could be simple denaturation (by acidicity) by the
buffer. Therefore, buffers (with a varying pH) were made from pH
2.0 to 12.0 and 1 ml of each was added to 1 ml of a chlorosome
stock solution. Absorbance spectra as well as R.sub.h were measured
for each sample. The R.sub.h was measured by testing 20 .mu.l of
the sample in the DLS system.
[0089] Heat (or temperature) induced photodegradation or
denaturation was also explored and tested. Starting at room
temperature, a water bath holding a vial of chlorosomes was brought
to near boiling over a period of hours. During the experiment,
absorbance readings were taken at about every 5-10.degree. C. and
degradation was calculated as mentioned previously.
[0090] Another mode of destruction of the photo-stability was
tested by increasing the concentration of the chlorosomes, in
solution to see if concentration aggregation could be attained.
This was accomplished by use of concentration filters and measured
by R.sub.h. 15 ml of sample was concentrated down to differing
volumes and the filtrate (buffer) was removed leaving a more
concentrated sample. Then 20 .mu.m of sample was removed for DLS
measurements after absorbance spectra were taken to ensure viable
sample and perform chlorosome counting.
[0091] A final experiment to show another mode of destruction of
photo-stability was the addition of a competitor (for absorbance of
blue light). Carotenoid solutions from the isolation procedure were
reintroduced into the chlorosome sample (by dilution) and emission
measurements were taken. Side control experiments were performed by
addition of buffer alone. Stimulation was made by the RF-1501
Shimadzu spectrofluorometer and emission was measured on a
photodiode after passage through an 800 nm interference filter (so
that scatter and excitation energies could be removed). This also
allowed for ratios of the Bchl c to a, Soret, and carotenoid peak
to be calculated and compared for potential enhancement
calculations for the hybrid well experiments.
[0092] The initial step in manufacture of the hybrid wells is the
chlorosomes (or controls) themselves. First the isolated solution
of chlorosomes had to be measured in order to determine the actual
number of chlorosomes (per ml) in the solution. For the controls,
this was done by number of molecules based upon molecular weight
(fluorescein) or counts supplied by manufacturers (unlabeled and
labeled particles). Next, calculations (and dilutions) had to be
made in order to develop a varying percent coverage. When all
coverages (and dilutions) were made, the procedure was the same.
Place 20 .mu.l of sample onto a hydrophobic coverglass and incubate
in the laminar flow hood, in the dark, for at least 10 minutes.
This gives the chlorosomes (or controls) enough time to physically
adsorb onto the borosilicate glass surface. Next, invert the
coverglass and place on top of the microslide holder centered on
the frosted ring (1 cm in diameter). Sealing is performed by use of
a 2-part (optical grade) epoxy. The samples are then placed in a
microslide holder and stored over night (at least 24 hours) in the
dark at room temperature. Further storage should be done at
5.degree. C. in the dark.
[0093] The biophotonic hybrid device had to then be assembled,
using the various interfacing techniques to integrate the
chlorosomes (and controls), in a controlled, patterned array with
the silicon (Si) photovoltaic (PV) photocell. Once fabricated, the
device parameters or specifications had to be tested. These
include: maximum output, time-response (or rise time), spectral
sensitivity, intensity sensitivity, temperature sensitivity, and
device lifetime.
[0094] The device was fabricated by utilizing physical adsorption
immobilization to interface chlorosomes (on a glass
substrate/microslide with well) to the Si PV photocell. The
components were interfaced (mechanically) by a self-built optical
chamber made from acrylic sheet. The microslide port was milled
into one piece, holes were drilled for the fiber optic bundle and
the Si PV detector. Accessory ports/chambers were made to fit 25 mm
filters such as additive (or subtractive) and NDF for wavelength
and intensity control, respectively. The whole apparatus was black
felted to reduce external light leakage. Power was supplied using a
standard variable power supply (for the LED) and the Si PV was
monitored utilizing a digital multimeter (DMM).
[0095] In this arrangement, device parameters such as maximum
output, time-response (or rise time), spectral sensitivity,
intensity sensitivity, temperature sensitivity, and device lifetime
were tested. Maximum output was monitored by allowing the device
sufficient time to go from 0 millivolts (mV) to maximum for that
particular LED intensity. The difference was recorded and compared
to when no sample is introduced. This ratio was defined as
normalized relative output in the following sections. Response time
is defined as the time that required going from 0 to 90% of the
final value during a switching on stage.
[0096] This was performed by timing a device versus the standard Si
PV detector (no device attached). Spectral sensitivity was
performed by replacing the LED with various colored LED's that
covered the visible spectrum as well as into the near infrared
(NIR). Voltages were recorded, and normalized relative outputs were
made on the final device. All devices were tested using 470 nm, 735
nm, 880 nm, and white LED's (full spectra). Intensity sensitivity
was verified using the blue (470 nm) LED since this was the
wavelength of choice for enhancement.
[0097] Stability was verified by absorbance spectroscopy (400-900
nm) and degradation was recorded. Temperature of operation was also
investigated and degradation was also monitored so that an
operational range of temperatures could be established. In these
experiments, data was also obtained to establish device lifetimes
under such `operational` conditions. The lifetime was determined by
seeing what conditions led to device degradation and the time
required to reach that point.
[0098] The devices (ranging from low to high percent coverages)
were tested under LED illumination and ratios were made to the same
detector under the same illumination (minus the hybrid wells) and
percentages have been calculated. This percent enhancement
signifies when a hybrid well device increases the measured output
(and by how much percent) over the stand-alone configuration. The
rise time (the time it takes to get from 10% to 90% final voltage)
was measured and compared between the hybrid well devices to the
stand-alone detector. This was accomplished with a stopwatch and
DMM. White light LED (visible light) stimulation of a series of
percent coverages have been conducted and compared to monochromatic
results. White LED's were implemented into the device apparatus and
run at a few intensities and on Si PV as well as Si TP. Device
enhancement was not further enhanced by the addition of the full
spectra light, in fact, red band quenching might have been recorded
as noted by others (Klar, 2000) in other systems and setups. Using
monochromatic light as the stimulation source intensities were run
at different levels sufficient for device detection but lower than
saturation (of device or hybrid component). Again, full range of
percent coverages were evaluated and replicates run. Again, the
detector's response (without the hybrid layer) was used as a point
of reference in determining percent enhancement.
[0099] Other tests included data gathered from solution effect
studies only. Temperature effects were determined by the previous
experiment in which the chlorosomes' photostability to temperature
changes was determined. Being in a bulk system in a water bath
controlled environment fitted the design parameters of the hybrid
well experiments and throughout testing, no experiments were
conducted outside the 25-100.degree. C. range. A series of hybrid
devices were constructed and tested (positively) over a large
period of time (and intensities). Further experimentation was
conducted on these samples throughout the research endeavor until
no (positive) responses were seen. Afterwards, the absorbance
spectra of a few high percent coverages were measured using a
home-built slide holder in the DU-65 Beckman Photospectrometer in
order to check the status (photostability) of the chlorosomes.
Forced Adaptation
[0100] Inconsistent results initially plagued experiments leading
to the development of the hybrid device of the invention. As noted,
C. aurantiacus is self-adapting. This meant that chlorosomes taken
from the same growth of cells could not be relied upon to behave
consistently.
[0101] To overcome this lack of consistency force-adapted C.
aurantiacus was developed having the performance desired. To this
end, and because of the number of environmental variables involved
in the growth of the cells and their substituent chlorosomes, a
design of experiment (DoE) technique was employed to arrive at
chlorosomes that performed well and consistently.
[0102] In relation to biophotonic device design, there are many
pertinent issues in each stage of the design. Variables encountered
throughout the entire process of making the hybrid photovoltaic
device that are capable of affecting results are set out in Table
2. The design of the product stage (as tested by validation
techniques) requires that the device be tested via an appropriate
light source type and wavelength (such as a 470 nm LED) or a
incandescent light bulb with correct interference filter (470 nm),
and can be projected to the surface of the device with or without
the aid of light pipes such as waveguides and/or fiber optics. The
intensity that the device is stimulated with must also be of
appropriate intensity as controlled by the voltage applied to the
source (LED for example), a neutral density filter (NDF), or other
means. Stimulation time must also be accounted for since the time
of stimulation and intensity will correlate to a certain
photostress that the device can handle before irreversible damage
is done to the biohybrid layer. A controlled environment (for
validation purposes) is also necessary (a dark room or constant
intensity area), as well as selection of an appropriate measuring
device, such as a high impedance digital multimeter (DMM) for
photovoltaic devices for example.
[0103] Before the device can be tested however, materials must be
acquired/produced and synthesized. These stages involve: growth of
the bacteria and alteration (if any) of the chlorosomes. In this
example, synthesis is governed by production in that changes in the
chlorosomes can be induced by the growth period factors. Some of
these factors include: intensity of light source, light type and
wavelength (incandescent, LED, fluorescent); media (pH,
temperature, components or strength); number of days allowed for
growth (before isolation or media exchange); bottle-fill volume;
and temperature. Some of these factors directly influence important
design characteristics such as Figure of Merit (FoM), chlorosome
size, photostability, and indirectly quenchers, to list a few.
[0104] Processing of the chlorosome requires isolation of the
chlorosomes from the whole cell walls. This is done using a
procedure well documented in the literature although certain
factors do arise in the process. There are different procedures
used to isolate chlorosomes without the reaction centers (RC.sup.-)
versus those with (RC.sup.+). The solvents, agents, and buffer
types used in the procedure are also very important and factors
such as (the type, molarity, ionic strength, pH, and strength) all
come into play. These factors will affect the state of aggregation
and purity (and successful use) of the isolated chlorosomes.
[0105] Manufacture of the chlorosome layer is the step whereby
means of immobilization (namely physical adsorption) a monolayer
(or percent thereof) is deposited onto the surface of a substrate
(borosilicate glass). Important factors for successful devices
include: the fabrication conditions (temperature, incubation time,
Light ON/OFF, and in the laminar flow hood); sealing method;
concentration, volume, and % coverage (and hence interparticle
distances); droplet placement (on the coverslip or in the well);
and coverslip hydrophobicity, which all relate to chlorosome
orientation (facing Si PV or LED).
[0106] Again, product final assembly is addressed above. However,
other issues pertaining to lifetime of device and/or other issues
such as post fabrication storage include factors of temperature,
light intensity (and quality--i.e. wavelength) and # days, to name
but a few.
TABLE-US-00002 TABLE 2 WELL ISSUES Growth Isolation Sample Fab
Sample Run Intensity Procedure Fab conditions Light Source type
Temperature LED Incubation time Light bulb Light ON/OFF W/wout F.O.
laminar hood Media Buffer Buffer Light Intensity Type Type
V.sub.applied Molarity Molarity NDF used Ionic Strength Ionic
Strength Measure tech. pH pH Stim. Time Temperature Temperature pH
Aggregated? Sealing method Light wavelength V.sub.applied LED Light
type Type (RC .+-.) Post fab storage Holder Incandescent
Temperature Sample holder LED Dark LED + NDF Fluorescent # days
SiPV + LPF Days of growth Purity % coverage Intensity control
Bottle Volume Volume Detector Temperature Droplet placed DMM used
On coverslip 9 V (new) In Well High Imped. Wavelength Coverslip
Orientation hydrophobicity Facing SiPV Facing LED Concentration Red
LPF used Voltage Applied NDF used Room lights ON/OFF Stimulation
time
[0107] In one stage of the development of the preferred exemplary
procedures described in the above example, a multiple input,
multiple output environmental chamber 150 (MIMO/EC) was constructed
as diagrammed in FIG. 22.
[0108] Nine compartments 155 through 163 were provided. Light
intensity increased from left to right across the three columns of
compartments and temperature increased from bottom to top across
the three rows of compartments. Within the compartments three
differing volumes of media were contained. Consequently, 27
combinations of variables were able to be tested. FIG. 23
illustrates an environmental chamber of this kind. On its door 170
multiple shelves 172 are supported and have openings to retain
culture-containing rest tubes or containers. Vertical dividers 174
separate the compartments 155-163. Horizontal dividers 176 separate
the compartments vertically. Light bulbs, one of which is shown at
177 provide illumination. A series of fans 179 regulate
temperature.
[0109] Ordinarily in biology research is conducted on the OFAT, one
factor at a time method. Here, the DOE approach permitted the three
factors, light intensity, temperature and media to air volume ratio
to be tested at three levels with three replicates and an
additional three centerpoints and a total of 27 experiments
(2.sup.3.times.3+3=27). The DOE technique allows for correlation of
data statistically easier than OFAT or best-guess approaches. It
reduces the total number of experiments, allows for a good,
thorough experimental design. It allows for error to be quantified
and it can distinguish if factors have any to no effect or if
interaction among factors occurs. Here, the response, the output
under study, was concentration (by absorbent spectroscopy) after
three days' growth.
Example
[0110] A factorial design was chosen to quantify the relative
importance of interaction between light intensity, temperature, and
volume of media. The approach used was Design of Experiments (DOE).
This method allows for data to be gathered in a way to avoid error
by establishing an experiment protocol and quantify error in a
mathematical way. The regression method that was used was the
analysis of variance (ANOVA) technique. This tool (DOE) allows for
data to be gathered at normal conditions (centerpoint) and at
extremes (above and below the centerpoint). Analysis is based on
quantifying effect and probability of effect of a factor or
interaction on the output variable.
Cell Culture Stock
[0111] The cell culture stock was prepared for testing as in [1]
and the MIMO/EC DOE culture incubation apparatus was also used. The
data was gathered from nine strands that all came from a
centerpoint grown stock (cultured at the centerpoint for 14 days).
The data was gathered (randomly) at the end of a three-day growth
cycle period and placed into the Stat-Ease.TM. software for
analysis. Three replicates at each corner were taken as well as
five centerpoint readings.
Pigment Protein Content Determination
[0112] The pigment protein content was deduced by taking absorbance
spectra from 650 nm to 900 nm on each sample. This was done with a
Beckman DU-65 photospectrometer. Then a ratio (R.sub.1) was
calculated by dividing the absorbance at 740 nm by that at 808 nm.
Then another ratio (R.sub.2) was calculated with the 740 over 866
nm peak absorbance readings. In this experiment, pigment protein
content was desired to see an increase (larger chlorosomes).
Statistical Analysis Approach
[0113] The DOE approach used involves seven steps in order to
perform the experiment. The first step involves defining the
problem statement. Here it was desired to investigate which factors
could increase the pigment protein content of the chlorosomes.
Next, the choice of the factors, which may influence pigment
protein content, had to be chosen. Also, the levels of these
factors had to be established. The factors that were chosen, and
their levels can be found in Table 3. below.
TABLE-US-00003 TABLE 3 Low, Centerpoint, and High Factor Levels for
DOE Experiment Factor Low (-) Center High (+) A = Temp 36.degree.
C. 48.degree. C. 60.degree. C. B = Intensity 50 lumen 270 lumen 490
lumen C = Media 5 ml 7.5 ml 10 ml
[0114] The next step is to identify the output variable(s) to be
studied. Since the change in pigment protein content was desired to
be analyzed, the ratios of the 740 to 808 and 740 to 866 nm peak
absorbances were chosen. The ratios were designated with a R.sub.1
for the 740/808 and a R.sub.2 for the 740/866 ratio. Since the
choice of factors and levels were as stated, a 2.sup.3 factorial
approach was chosen. In this approach, three replicates and five
centerpoints were chosen also. The experiment was run at the end of
a three day growth period and data was gathered in a random
fashion. Since replicates were used the data analysis will not
include determination normal % distribution plot and the analysis
will really be based on the ANOVA tables. Interaction between
factors was determined from the ANOVA as well as the interaction
graphs provided by the software. Finally conclusions must be made
based on the analysis and results.
[0115] The R.sub.1 ratio developed strong effects due to each
individual factor and the interaction between Temperature and %
Volume. All other interactions were insignificant when compared to
these four factors/interaction. This can be seen in the ANOVA table
in Table 4. The normal % probability plot and interaction plot
(between Temp and % Vol) can be found in FIGS. 11, 13a and 13b.
Based on the analysis, the highest level for the R.sub.1 ratio
would be with bacteria grown under the following conditions: low
temperature, low light intensity, and high % volume.
TABLE-US-00004 TABLE 4 ANOVA Table for experiment. Factor CE DF
Error Prob > |t| Intercept 1.23 1 9.913 .times. 10.sup.-3
A-Temperature -.032 1 9.913 .times. 10.sup.-3 .0041 B-Light
Intensity -.037 1 9.913 .times. 10.sup.-3 .0013 C-% Volume -.044 1
9.913 .times. 10.sup.-3 .0003 AB .027 1 9.913 .times. 10.sup.-3
.0118 AC -.056 1 9.913 .times. 10.sup.-3 <.0001 BC .015 1 9.913
.times. 10.sup.-3 .1372 ABC -.021 1 9.913 .times. 10.sup.-3 .0502
Centerpoint .41 1 .024 <.0001 Note DF represents degrees of
freedom and CE is coefficient estimate. An appropriate prob >
|t| was chosen to be 0.01 for this output variable therefore A, B,
C, and AC have an effect on this output.
[0116] The R.sub.2 ratio developed strong effects due to only
temperature and no interactions. All other factors and interactions
were insignificant when compared to temperature (see Table 5). The
normal % probability plot and interaction plot (between Temp and %
Vol) can be found in FIGS. 13a and 13b. As shown in FIG. 14a, the
results are so close to the linear line that they are deemed
insignificant except for temperature. Even the interaction plots
(FIG. 14b) showed slight interactions. Note how the lines cross but
the error bars overlap so that these lines could in fact be
parallel and therefore non-interacting. The highest level possible
for the R.sub.2 ratio would be with bacteria grown under low
temperature.
TABLE-US-00005 TABLE 5 ANOVA Table for experiment of R.sub.2 ratio.
Coefficient Factor Estimate DF Error Prob > |t| Intercept 1.23 1
.047 A-Temperature -.11 1 .047 .0321 B-Light Intensity .032 1 .047
.5080 C-% Volume .006 1 .047 .8990 AB .049 1 .047 .3050 AC -.035 1
.047 .4651 BC -.036 1 .047 .4516 ABC -.077 1 .047 .1181 Centerpoint
.51 1 .11 .0002 Note DF represents degrees of freedom. An
appropriate prob > |t| was chosen to be 0.1 for this output
variable therefore A, B, C, and AC have an effect on this
output.
[0117] It is interesting to note from the results that the response
variables (namely R.sub.1 and R.sub.2) are not dependent upon the
same factors. R.sub.1 is sensitive to temperature, light intensity,
and % volume and the interaction of temperature and % volume.
However, the R.sub.2 ratio is dependent upon only the temperature
during growth. This ratio was long believed to be only dependent
upon light intensity but temperature was more significant. This may
be due to the fact that the real dependent output is the R.sub.1
ratio. If the bacteria are grown under those conditions and R.sub.1
changes, R.sub.2 must change as well but not vice-versa.
[0118] It is also possible that the temperature affects only the
866 nm molecules and light never changes growth (within limits
selected in this study). Another, stronger argument is that since
the natural funnel-like energy transfer in the chlorosome (from 740
to 795 to 808 to 866 nm molecules) protects the molecules further
down the chain (like the 866 Bchl a) from being sensitive to
factors such as light. At the same time, these molecules are still
protein based and very dependent upon temperature effects.
TABLE-US-00006 TABLE 6 DOE experiment calculated data for
pigment-protein growth/development ratios over a period of 6
transfers (3 weeks approximately). condition ratio Nov. 18, 1997
Nov. 21, 1997 Nov. 25, 1997 Nov. 28, 1997 Dec. 2, 1997 Dec. 9, 1997
--- 740/808 1.3182 1.2 1.1111 1.2174 1.25 1.0526 740/866 1.45
1.3333 1.1111 1.4 2 1.3333 --+ 740/808 1.2381 1.1071 1.16 1.28
1.2308 1.1667 740/866 1.3929 1.1273 1.2889 1.4545 1.4545 1.25 -+-
740/808 1.1579 1.0345 .9231 1.1842 1.0526 1.017 740/866 1.2571
1.0526 .8571 1.2857 1.0909 1.3 -++ 740/808 1.2353 1.375 1.2027
1.1667 1.2 1.4 740/866 1.377 1.5068 1.3692 1.3462 1.3548 1.4848 +--
740/808 1.3333 1.2222 2.0732 2.1458 1.88 1.125 740/866 1.4545 1.375
2.2667 2.4235 2.0435 1.1538 +-+ 740/808 1.6 1 1 2.3368 1.7412
1.9759 740/866 2 1 1 2.6118 1.9221 2.2162 ++- 740/808 1.2857 1.0952
1.1579 1.2917 1.2687 1.25 740/866 1.4062 1.2778 1.2941 1.4531
1.4167 1.3514 +++ 740/808 1.1111 1 1 1.3049 1.2273 1.3929 740/866
1.3333 1 1 1.4079 1.35 1.56 000 740/808 1.7105 1.7901 1.7901 1.5854
1.686 1.5224 740/866 1.8571 1.9079 1.9595 1.6667 1.7262 1.619
[0119] Changes could clearly be seen from one transfer to the next.
This suggests a forced evolution situation. The bacteria are being
forced to survive in a hostile environment.
[0120] Take the +-- bacteria. In the first two transfers, it looked
like it was dying and then by the third environment, some cells
have adapted to the different environment and grown. Remember that
the +-- was high temperature, low light intensity, and low amount
of food source. Other changes can be seen in this sequence of
pictures but clearly, this was the most significant.
[0121] The light intensity and the light-temperature interaction
factors had coefficients of only one half the temperature factor in
the 740 nm variable. This contrast was particularly apparent in
those response variables that do not have photosynthetic activity.
There is clearly a correlation between the light factor, the light
and temperature interaction, and the absorbance of Bchl c (740 nm).
Since the other response variables are mostly dependent on
temperature, their changes can be primarily attributed to the
change in absorbance which results from increased and/or decreased
concentration of cells. Because cellular membrane components have
an absorbance of 650-700 nm, the concentration of cells in each
sample can be determined from the absorbance data in this region.
By normalizing the data, it is possible to extrapolate the Bchl c
absorbance for individual cells. This is the next logical step in
analyzing the data.
[0122] A method was developed to establish a faster process to
count whole cells. A modified hemocytometry counting technique was
used to count whole cell C. aurantiacus concentrations (per unit
length of 10 .mu.m), and absorbance data was gathered as three
replicates of: 1:1, 1:1.1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:10, and
1:50 dilutions were made. Full spectra (absorbance) data was
gathered for each dilution, as in FIG. 15a. Each replicate was run
to minimize instrument and operator error, FIG. 15b and peak data
was gathered and averaged at 650, 740, 808, and 866 nm. The samples
were then counted on an optical microscope using a standard red
blood cell counting technique and a hemocytometer. In this fashion,
curves were developed for absorbance at 650 and 740 nm and cellular
counts with error bars as in FIGS. 16a and 16b.
Counting Chlorosomes
[0123] For the purposes of characterization and conformity in
preparing the hybrid devices contemplated, determining the quantity
of chlorosomes coating the cover glass hydrophobic surface was
important. Absorbance of light was correlated to the density of
chlorosomes as illustrated in FIG. 17. The calibration plot of FIG.
17 plots chlorosome count against chlorosome absorbance at the 650
nm wavelength. The 650 nm wavelength is chosen rather than a
wavelength where absorbance of the chlorosome exhibits a peak
because the absorbance at those wavelengths exhibiting a peak in
the absorbance spectrum vary from one chlorosome to another
depending, inter alia, on environmental factors effecting the
growth of the bacterium from which the chlorosome was taken. The
650 nm wavelength absorbance, then, is linearly related to
chlorosome count and not another variable.
[0124] In the exemplary preferred embodiment employing the
chlorosomes of C. aurantiacus to enhance SiPV performance,
chlorosome percent coverage of the SiPV's light receiving surface
(or the overlying borosilicate glass) is important as demonstrated
by the FIG. 19 plot of percent enhancement against percent
coverage. Ideally, in this particular embodiment at least, coverage
should be in the 4 to 7% range and preferably about 4%.
[0125] To arrive at percent coverage, accurate counting of the
chlorosomes becomes important.
[0126] FIG. 1 is a conceptual block diagram that indicates the
design and development of a hybrid device of the nature of the
enhanced photovoltaic cell described above. At each stage of
development multiple variables entered the design process. This is
tabulated, as well, in Table 2. From this it will be seen that a
robust program such as the design of experiments program that
permits the assessment of multiple variables and their interaction
is an enabling design tool in arriving at a final product that
meets the objectives of high performance, robustness, scalability,
energy interactivity and adaptability.
[0127] In the case of the enhanced hybrid photovoltaic device many
pertinent issues arise at each stage of design. At the product
stage, the device needs to be tested using an appropriate light
source and wavelength, such as a 470 nm LED or an incandescent
light bulb with a correct interference filter yielding 470 nm
wavelength. Intensity is a variable. The use of suitable light
waveguides or fiber optics may be a variable to consider.
Stimulation time must be taken into account since it and intensity
will correlate to a certain photostress that the device will be
able to handle or not handle if irreversible damage is to be
avoided. Controlled environments and appropriate measurement
devices are to be chosen.
[0128] Before the device can be tested, however, materials must be
acquired, and/or produced as by systhesization. Here the stages
involve growth of the bacteria and alteration if that is needed to
alter the chlorosomes that are to be employed. As has been seen,
factors involved in the growth period can affect the chlorosomes,
either beneficially or not. Some of these factors include light
intensity, light type (i.e. incandescent, LED or fluorescent) and
wavelength. Media (its PH, temperature, components, and strength)
can affect chlorosome yielding bacteria development. The number of
days allowed for bacterial growth (either before isolation of the
bacteria or before an exchange of media) is another factor.
Bottle-filled volume or, as has been shown, percent media to air
and temperature are factors, as well. Some of these factors
directly influence important design characteristics such as "Figure
of Merit," discussed below, chlorosome size, chlorosome
photostability, indirect quenching, etc.
[0129] Processing of the chlorosome requires isolation of the
chlorosomes from the whole cells. As indicated above, this is done
using procedures well documented. Nevertheless certain factors need
to be taken into account during this process. These are the
different procedures used to isolate chlorosomes without the
reaction centers (i.e. the RC.sup.- chlorosomes vs. the RC.sup.+
chlorosomes). Solvents, agents and buffer types used in the
procedure are also important, and factors such as the type,
molarity, ionic strength, pH and strength of these all come into
play. These factors will affect the state of aggregation impurity
of the isolated chlorosomes, and consequently the ultimate success
of the design.
[0130] Manufacture of the chlorosomes layer is the step whereby
means of immobilization (which is to say physical absorption) of a
monolayer (or a percent of a monolayer) is deposited onto the
surface of the substrate such as the borosilicate glass. Here,
important factors for successful devices include the fabrication
conditions of temperature, incubation time, lighting (on or off)
and operation of a laminar flow hood. Sealing method, concentration
volume and percentage of coverage enhance interpartical distances,
dropment placement on the cover slip or in the well, and cover slip
hydrophobisity all bear on chlorosome placement and orientation
(i.e. either facing the SiPV or the LED in the preceding exemplary
arrangements).
[0131] Final product assembly involves many of these same factors
just raised, others described above and other factors commonly
encountered in product production. Further concerns relate to
device lifetime, postfabrication storage including temperature
light intensity, type and wavelength are additional concerns.
[0132] From the above, then, it should be evident that best guess
or one factor at a time approaches to design and development pale
in comparison to the DOE approach.
Figure of Merit
[0133] "Figure of Merit" (FoM) is a concept employed widely and in
many disciplines, although ordinarily not where biological matters
arise. In the present invention a biophotonic Figure of Merit was
devised to quantify chlorosome performance.
[0134] Bearing in mind the chlorosome functioning as conceptually
diagrammed in the block diagram of FIG. 21a, the following Figure
of Merit was devised.
F o M = % T 440 ( Bchl c Soret ) % T 440 ( Bchl c Soret ) + % T 460
( Carotenoid ) * % T 795 ( Bchl a Baseplate ) % T 740 ( Bchl c
Oligomeric Qy ) . ##EQU00001##
[0135] This FoM takes into account the total transmittance of the
Bchl c Soret at 440 nm as compared to the total Soret and
corrotenoid 460 nm transmittance and the baseplate Bchl a
transmittance at 795 nm as compared to the Bchl c oligomeric
transmission at 740 nm.
[0136] Engineering to a Figure of Merit in the exemplary embodiment
of this invention was calculated to yield 160% of the Vout response
of the original silicon photovoltaic cell at a Figure of Merit of
1.0. The actual improved output was measured at 157%.
[0137] Although preferred embodiments of the invention have been
described in detail, it will be readily appreciated by those
skilled in the art that further modifications, alterations and
additions to the invention embodiments disclosed may be made
without departure from the spirit and scope of the invention as set
forth in the appended claims.
* * * * *