CN100404122C - photocurrent generator - Google Patents

Abstract

The invention provides systems having an electron transfer moiety tethered to an electrode by a conductive spacer moiety. A biasing potential applied to the electrode reduces the electron transfer moiety to form a reduced electron transfer species capable of absorbing a photon, to form an excited electron transfer species. An electron accepting moiety accepts an electron from the excited electron transfer species, to form a reduced electron acceptor. The reduced electron acceptor may for example be used in hydrogen generation reactions.

Description

Photocurrent generator

Invention field

The invention belongs to photochemistry current generating apparatus field.

Background of invention

The surface of existing multiple golden modification is used to produce and analyze photoelectric current [7-9].By ITO or Au large electrode (macroelectrode), multiple photon can be received group, or moiety combinations is applied to photocurrent generator, fullerene for example, [6,8,11-32] porphyrin, [5,6,8,9,11,13-16,20,21,23-25,29-31,33-44] ferrocene, [5,8,13,23,24,29,36,42,45] Ru (bipy) ₃[29,46-48] and pyrene [7-9,45].In some cases, the generation of photoelectric current is by biomolecule group mediation [7,49-52] at interval.

Summary of the invention

Aspect selectable, the invention provides the system that comprises such as the electron transfer moiety of the reception photon of fluorescein, wherein this electron transfer moiety is connected to electrode (it can be the arbitrary surfaces that can carry out the electronics transduction, i.e. chemotron) by the conductibility compartment such as nucleic acid.Apply bias potential reducing the electron transfer moiety of described reception photon to described electrode, thereby form the electronics transfer of material of the reception photon of reduction, FI-free radical for example, it can absorb photon and form the electrons excited transfer of material.Described system also has the electronics receiving unit, for example NAD or NADP, and it can receive electronics from described electrons excited transfer of material, thereby forms the electron acceptor of reduction, for example NADH or NADPH.Described electronics receiving unit can place and contain the electrolytical solution of supporting that electronics shifts, and this solution can be described as electronics and shifts solution, and the aqueous solution of proton for example can be provided to the electron acceptor of described reduction.The electron transfer moiety of described connection can be immersed described electronics and shift in the solution, thereby supply the described electrons excited transfer of material in the described solution and the electron transfer reaction of the repetition between the continuous electronics receiving unit.Can select the used electrochemical substance of this system, make that put on the bias potential that described electrode is used for forming the electronics transfer of material of described reduction is lower than the required electromotive force of electron acceptor that forms described reduction, thereby make the tendency that electron transfer reaction forms the electron acceptor of described reduction does not take place on described electrode.Can select the assembly of described system, thereby make the generating rate of electronics transfer of material of described reduction be higher than described electrons excited transfer of material is supplied with speed from electronics to described electron acceptor, thereby make when when described electrode applies suitable bias potential, most of described electronics transfer of material is in goes back ortho states, and it is easy to absorb photon and forms described electrons excited transfer of material.

The electron acceptor of described reduction can be used for for example producing the reaction of hydrogen.

In some embodiments of the present invention, can shift to add in the solution to described electronics and can utilize the electron acceptor of described reduction such as enzyme or other chemistry or the biochemical system of NAD (P) H, utilize the electron acceptor of described reduction.In these embodiments, the electron acceptor of described reduction can be that bioactive enzyme cofactor is for example arranged.The co-factor that described Optical Electro-Chemistry produces for example can carry out that zymetology is used to drive the conversion of aldehyde to alcohol, the reduction of ketone, reductive amination or organic acid reduction.Therefore, the co-factor of photochemical regeneration of the present invention such as NAD (P) H can be used for driving multiple secondary living things catalysis and transforms, and for example reduction transforms or the enzyme cascade of living things catalysis.

Brief description of drawings

Figure 1 shows that the schematic diagram (as described herein, alternative embodiment can be used multiple electrode structure and surface type) that uses microelectrode to produce the experimental provision of photoelectric current.

Figure 2 shows that (a) in KOH, and (b) under the condition that KOH and fluorescein exist, at pH12,50mVs ^-1The time dark (Dark) electric current CVs on the BAS large electrode.(b) at pH8.6,50mVs ^-1The time turn on light (-) and the turn off the light CVs of microelectrode under (--) condition.Reference electrode is Ag/AgCl.

Figure 3 shows that (a) applies duration (vs.Ag/AgCI) i at different electromotive forces) 0mV, ii)-and 750mV, 1min, iii)-750mV, 2min, iv)-750mV, 3min, v)-750mV, 6min, vi)-750mV, 10min, vii)-and 750mV, the UV-visible absorption spectra of fluorescein under the condition of 20min; (b) apply duration (vs.Ag/AgCl) i at different electromotive forces) 0mV, ii)-and 750mV, 1min, iii)-and 750mV, 2min, iv)-and 750mV, 3min, v)-and 750mV, the emission spectrum of fluorescein under the condition of 4min.

Figure 4 shows that the ERP spectrum of (a) 1:2 after a large amount of electrolysis of-750mV (vs.Ag/AgCl) 1 hour; And the ERP spectrum of the 1:2 that (b) simulates.

Figure 5 shows that wherein being depicted as in (a) solution has NADP from the data that produce the embodiment of photoelectric current by the 1:2 individual layer on the Au microelectrode ⁺(b) there is not NADP in the solution ⁺And (c) 632nm radiation (power=10mWcm ^-2) irradiation under the 1:2 individual layer and solution in have NADP ⁺

Figure 6 shows that and lacking NADP ⁺(mouth) and have NADP ⁺(O) under the situation, (a) photocurrent response is to the function of the reduction potential that applied; And (b) photocurrent response to the function of luminous intensity.

Fig. 7 has shown repeatedly excitation response, and it shows that photoelectric current has small size reduction as the function of number of repetition.

Figure 8 shows that at 473nm radiation, 4mWcm ^-2With 0.1mM NADP ⁺Under the condition of solution, from the spectroelectrochemistry data of the 1:2 individual layer on the Au net electrode (mesh electrode).(a) 0mV (-) and-baseline NADP under 750mV (--) the vs.Ag/AgCl condition ⁺(b) be the UV-visible spectrum that lactic dehydrogenase and pyruvic acid add preceding (--) and adding back (-).

Figure 9 shows that photoelectric current produces and NADP ⁺The schematic diagram of inferring mechanism of reduction, it is only for concept nature purpose (and needn't describe the actual mechanism that embodiment of the present invention operates).

Figure 10 shows that the schematic diagram of hydrogen generator of the present invention, wherein the dark reaction cabin contain the electron acceptor " NXH " that can utilize reduction (for example NADH[we can insert the description of selectable nicotinamide derivates, if you know that they are and so on words? ]) hydrogenase or other catalyst synthesize H ₂The electron acceptor NXH of wherein said reduction is provided by light reaction of the present invention, this light reaction with light reaction cabin that described dark reaction cabin exists liquid to be connected in carry out, the photon receptor (fluorogen " F ") that wherein is connected to electrode (electrochemistry conductive surface) has mediated the synthetic of described NXH.

Figure 11 shows that the schematic diagram of the UV-tangible proof that the photoinduced NADH electrochemistry on the golden net electrode of 1:2 modification is synthesized.

Figure 12 shows that under the condition that acetaldehyde (acetylaldehyde) exists, show by alcohol dehydrogenase (ADH, Baker ' s Yeast, the schematic diagram of the UV-visible spectrum that the NADH enzyme that Sigma-Aldrich) causes consumes.

Figure 13 shows that the schematic diagram of inferring mechanism of the NADH photogenerated effect on the self-assembled monolayer of the fluorescein-labeled DNA on the gold electrode, it only is concept nature purpose (needn't describe the actual mechanism of embodiment of the present invention running).

Figure 14 a-b is depicted as described self-assembled monolayer on the gold electrode generates photoelectric current under radiation schematic diagram.(a) 473nm contains NAD ⁺(b) 473nm does not have NAD ⁺632nm contains NAD ⁺Scale (scaler) is Y=200nAcm ^-2, X=20 second.

Figure 15 a-b is depicted as (a) NAD ⁺(O) and do not have a NAD ⁺Under the condition (), current density is to the function of described incident intensity, and (b) current density to the function of the electromotive force that applied.

Figure 16 a-b is depicted as (a) from NAD ⁺Form the spectrophotometric analysis of NADH photogenerated effect.The formation of the corresponding NADH in the peak of 340nm.It is 5 minutes radiation that every curve is represented step-length.The volume of cuvette is 0.12ml.And the catalysis of the alcohol dehydrogenase (0.5U/ml) that (b) relies on by NADH-, utilize NADH to drive the conversion of acetaldehyde (10mM) to ethanol.The corresponding NADH of the reduction of 340nm place absorbance is to NAD ⁺Conversion.Every curve has been represented 3 minutes step-lengths.Under the situation that lacks acetaldehyde or alcohol dehydrogenase, the spectrum no change.

Detailed description of the invention

On the one hand, the self-assembled monolayer (SAM) that the invention provides the fluorescein-labelled DNA from the golden microelectrode produces the system of photoelectric current. In this class embodiment, fluorescein is as photon receptor (or fluorogen), and DNA is as the interval group that described photon receptor or fluorogen is connected to described electrode surface. Fluorescein has relatively large molar absorptivity, thereby easier absorption photon carries out subsequent reactions [10]. In exemplary embodiment, the partly cause that uses described DNA interval group is that dependent its photoelectric current of short interval group that studies confirm that of internode length reduces, and shows by the close proximity electrode surface to make its inactivation of fluorogen with excited state. In the situation that adapts to these restrictions, the present invention also can select other fluorogen and interval group. Selectable interval group can comprise, conductive polymer for example, and such as the Polypyrrole class, polythiophene class, polyphenylacetylene class, peptide class, polyamide or peptide nucleic acid (PNAs). Selectable photon receptor comprises porphyrin, flavin, ubiquinone class, quinones, ferrocene, Ru (bipy)₃, methylene blue, methylene is green, MV⁺, pyrene and nano particle class (for example Au, Ag, CdSe, SdS, ZnSe, ZnS, Pd, Pt). Selectable matrix can comprise, for example, indium tin oxide (ITO), Ag, Pt and Si surface, it can form the surface with various topological structures, from microelectrode to large plane surface. The ITO electrode group (electrode stack) of substantially transparent is applicable to the electric current that passes electron acceptor is provided, thereby described electron acceptor (for example NAD (P) H) enters from the shadow surface of described electrode group, then from the leaving without shadow surface of described electrode group, the electrode group of substantially transparent described in this process can both be assisted the irradiation to described system to the electron acceptor (for example NAD (P) H) of reduction in the entire depth scope of described electrode group.

As shown in figure 10, the electron acceptor of described reduction can be used for for example producing in the reaction of hydrogen. Such as Figure 11 and shown in Figure 12, the NADH that generates by system of the present invention can be used for enzymatic.

Figure 11 has shown that the photoinduced NADH electrochemistry on the golden net electrode of 1:2 modification produces.

Figure 12 has shown that acetaldehyde exists down, by alcohol dehydrogenase (ADH, Saccharomyces cerevisiae, the enzyme consumption of the NADH that Sigma-Aldrich) causes.

In another embodiment of the present invention, shift the enzyme biochemistry system that has added the electron acceptor NADH that utilizes described reduction in the solution at described electronics, the utilization to the electron acceptor of reduction with BA has been described.Shown in Figure 16 b, the NADH that Optical Electro-Chemistry produces can be used to drive acetaldehyde and transform to the zymetology of ethanol.Under certain conditions, described process is to oxygen, and the inhibitory action of organic solvent and other compounds has tolerance.In the embodiment of replacing, the electron acceptor such as the reduction of NAD (P) H that is produced by system of the present invention can be used for multiple other reactions.

Embodiment 1

Material and preparation

At the National Research Council (Saskatoon, SK, the DNA that Canada) adopted the synthetic and purifying of standard DNA synthetic method, and its purity and characteristic verified.By being fixed on the 50 μ m Au silks fusing in the soft glass (soft glass), with the polishing of 0.05 μ m aluminium oxide slurries, immerse the Piranha etch solution (H of heat more then ₂SO ₄: H ₂O ₂=3: washed (operation Piranha etch solution should be extremely careful, and it can not be stored in the closed container, and it is very strong oxidant, can with most of organic material vigorous reactions) 1) in 10 minutes, at last at Millipore H ₂Carry out ultrasonic wave among the O and handle, prepare gold electrode.Adopt light microscope that each electrode is checked, smooth to guarantee described Au electrode surface, and between described glass and described Au, formed effective seal.Then at 0.5M H ₂SO ₄By scan round described electrode is carried out electrochemical treatments in the solution, up to obtain stable golden oxidation peak at 1.1V from electromotive force-0.1 to+1.25V vs.Ag/AgCl.

By described microelectrode is being contained the 50mM Tris-ClO of 0.05mM double-stranded DNA ₄Cushioning liquid (pH8.6) in hatched 5 days, prepared the gold electrode of FI-DNA modification.Then, as shown in Figure 1, with described electrode with identical Tris-ClO ₄Cushioning liquid cleans, and in equipment light inlet-electrochemical cell.In order to get rid of the counterelectrode reaction that may influence chronoamperometry, preferably described counterelectrode is isolated.

The photoelectric current condition is as follows, is 4mWcm with laser power ^-2, wavelength is 473 ± 5nm and beam diameter (Intelite Inc.Genoa, NV is USA) as excitaton source less than the BM73-4V laser module of 0.8mm.The experiment of described photoelectric current is to carry out under employing is connected to the voltage clamp condition of Axopatch 200B amplifier (Axon Instruments) of CV 203BUheadstage.The configuration of two electrodes is used for the voltage clamp condition, and reference electrode is the Ag/AgCl silk in the 1M KCl solution, and working electrode is the Au microelectrode of modification.Described spectroelectrochemistry battery is packed in the faraday's cage (Warner Instruments) of ground connection, and is positioned on activation air vibration proof (Kinetic System) platform.Electric current passes through the filtering of low pass Bezier at 1kHz, and carries out digitlization at 5kHz by DigiData 1322A (Axon Instruments), and by PClamp 9.0 (Axon Instruments) record by PC control.Further filtering is to adopt the low pass filter of 20Hz to realize by software approach.Analysis to all data is to use Origin7.0 (OriginLab Corporation) to carry out.Other electrochemical measurement is to use the BASCV-50 voltmeter and carries out for the electro-chemical systems of the microelectrode customization of employing standard 3 electrodes configurations.Described golden microelectrode (50 μ m diameter) can be used as working electrode.Have in the glass tube of Vycor tip (tip) by the Ag/AgCl silk being encapsulated into contain 3M KCl and add a cover (capped), made up reference electrode.By containing described electrolytical Luggin capillary (Luggin capillary) described reference electrode is isolated from described battery.Described counterelectrode is a platinum filament.Before measuring, whole electrolyte solutions must be purified (purge) at least 20 minutes in air, and in measuring process, on described solution, keep being covered with Ar atmosphere.All embodiments are all exemplarily at room temperature carried out.

The x-ray photoelectron spectroscopy method is implemented as follows.To be equipped with the Leybold MAX200 photoelectric tube spectrometer of Al-K á radiation source (1486.6eV) to be used to collect photoelectron emissions spectrum.In measuring process, the base pressure in described analysis cabin remains at least 10 always ^-9Mbar.The described angle of emergence is 60 °.Conventional instrumental correction standard is Au 4f7/2 peak (binding energy 84.0eV).

The enforcement of electron paramagnetic resonance (EPR) is as follows.With the described EPR spectrum of Bruker ESP300 X-band field scan spectrometer (the about 9.4GHz of resonant frequency) record that has been equipped with high sensitivity cylindrical cavity (Model4107WZ, Bruker Spectrospin).Modulation amplitude is 0.315G, and microwave power is 20mW, and transformation time is 41ms, and time constant is 20.5ms, and has write down 32 scanning.Use SimFonia software to carry out the simulation of EPR spectrum.

Result and discussion

The synthetic standard phosphoramidate solid support that is to use of described fluorescein-labeled DNA (FI-DNA) is at NRC, the Saskatoon, and Canada finishes.The sequence that is used for described photoelectric current experiment is as shown in table 1.Described base sequence is selected, minimizing optional secondary structure or tertiary structure, and introduced every kind of base of equivalent.Carry out DNA fusion research and confirm the existence/shortage of described double chain form, and guarantee that the fluorogen of described fluorescein can not cause appreciable impact to the stability of two strands.The two strands that the DNA melting curve of 1:2 two strands is compared 2:3 illustrates that described fluorescein does not partly significantly disturb double-stranded formation on the Tm value and no change (56.8 ℃ vs.56.4 ℃).

Table 1 is used for the dna sequence dna FI=fluorescein of photoelectric current research

1	HO-(CH 2) 6-S-S-(CH 2) 6-5’-GTCACGATGGCCCAGTAGTT-3’-FI
		2	5’-AACTACTGGGCCATCGTGAC-3’
3	HO-(CH 2) 6-S-S-(CH 2) 6-5’-GTCACGATGGCCCAGTAGTT-3’

Described 1:2 is double-stranded hatched in buffer solution 5 days with the Au microelectrode, made to form complete individual layer.Adopt x-ray photoelectron spectroscopy method (XPS), ellipsometry and electrochemical process are analyzed individual layer.The variation of Au4f7/2 peak intensity can be used to measure the thickness of described individual layer, and has provided 47 (5)

Value, its explanation 1:2 can not form sandwich construction.As desired to the 1:2 individual layer, the S2p peak that occurs at 162eV is the evidence of Au-mercaptan key.Disulfide bond that it should be noted that expection 1:2 can rupture to described Au surface by chemisorbed, and does not observe the energy peak (164.1eV) of disulfide bond.In addition, at 134eV the P2p peak is measured, it is corresponding to the phosphate backbone of DNA.Described XPS result provides evidence clearly, illustrates that individual layer connects described Au surface by sulfide linkage.The thickness that ellipsometry obtains the 1:2 individual layer on the Au matrix is 47 (3)

The measurement [53] of this numerical value and 20-mer DNA before coincide, and consistent by the numerical value that XPS obtains with self, illustrates that described DNA has sizable inclination angle to described surface.

Carry out electrochemical test and detect the redox potential of fluorescein with 1:2 individual layer.Yet, because the self-characteristic of described fluorescein dynamics of oxidation reduction makes cyclic voltammetry (CV) experiment complicated.Described electrochemical reduction/oxidation is too slow, analyzes and be difficult to carry out conventional CV.Shown in Fig. 2 a, also inequality under the situation of CV and shortage fluorescein although in the presence of fluorescein, but there is not recognizable reduction peak.Fig. 2 b is depicted as in the dark and the naked Au CV of fluorescein when being subjected to radiation.When described electrodes exposed during in radiation, can less variation take place towards the direction of higher positive current.Complicated situation is that described reduction potential is about-750mV vs.Ag/AgCl, and it is relatively near the proton reduction potential under the used pH condition.Therefore, because described dynamics of oxidation reduction slowly, and therefore formal potential can not have reduction peak clearly near the electromotive force that produces hydrogen.Its consequence is that the surface coverage value can not be as the redox probe with good behaviour is passed through electrochemical quantitative.The approximation of described surface coverage is to use identical dna double chain to obtain, except having replaced fluorescein with ferrocene (Fc).The surface coverage of this Fc individual layer it is reported to be 5 * 10 ^-10Molcm ^-2Utilization impedance spectroscopy (IS) is come these two kinds of individual layers of comparison (1:2 contrasts 1-Fc:2), thereby verifies whether described surface coverage approximation is effective.Clearly, described IS result shows the performance that has much at one under described the same terms, thereby described surface coverage value is judged to be within 20%.

Carry out the evidence that the fluorescein spectroelectrochemistry is tested provides the stimulative substance that is adopted in the actual light electric current generation experiment.Surpass when applying amplitude-during the electromotive force of 750mV, the light absorption of UV-visibility region demonstrates tangible change in the described spectrum.Described spectrum change as shown in Figure 3.The reduction of described fluorescein absworption peak (492nm) has been reduced into fluorescein anion (FI-) owing to described fluorescein (FI).FI-has unique spectrum, and is different with FI, and the peak that shows as 380-420nm and 550-650nm scope increases [55-57].

What follow described UV-visible spectrum variation is the reduction of described fluorescein fluorescence spectrum.The reduction of the fluorescein fluorescence intensity shown in Fig. 3 b illustrates that the FI-material has lower fluorescence quantum yield.The reduction of this inactivation approach may be to be caused by the growth that electronics shifts (ET) inactivation approach.

Electrochemistry EPR double-stranded to described 1:2 and fluorescein studies and confirmed to be higher than beyond all doubtly-and during the electromotive force of 750mV, the FI of described reduction is as fluorescein radical anion (FI ^-).

The EPR spectrum of described 1:2 and fluorescein and corresponding simulating spectrum thereof are as shown in Figure 4.Employed simulated spectra numerical value sees Table 2, and from prior art references [58-66].

The coupling constant and the unpaired spin density of table 2FI and FI-DNA proton

As shown in Figure 5, the radiation of 1:2 individual layer caused applying-during the electromotive force of 750mV, produced photoelectric current.When applying described negative potential, suppose to have the electron acceptor that is fit to, the then FI of described excitation state ^-Free radical becomes and can transmit electronics, thereby produces electric current.In this embodiment, with NADP ⁺Add in the described solution as acceptor groups.Importantly, in producing the essential electromotive force zone of photoelectric current, NADP ⁺Not by electrochemical reduction, so the NADP on the described electrode ⁺Electron acceptor also can not reduce.Lacking NADP ⁺Also observe photoelectric current under the situation of (Fig. 6 b), but had NADP ⁺(photoelectric current significantly strengthens under Fig. 6 situation a).Red laser (632nm, 10mWcm ^-2) radiation then can not produce photoelectric current (Fig. 6 c).

The dark reaction synthetic for photon, NADP ⁺Be important chemical energy deposit, therefore, can develop as the energy reserve in the biosystem.Fig. 6 a is depicted as by to the electric current that radiation produced of the described individual layer function to applying electromotive force.Described electric current reaches maximum when pact-750mV, then sharply descend at lower applying electrical potential.-the 750mV value is maximum to illustrate that described fluorescein is reduced into its free radical anion before radiation, carries out the electronics transmission then.Shown in Fig. 6 b, between described incident laser and output photoelectric current, there is linear relationship.

By being exposed to laser repeatedly the utilizability that described individual layer stands repeatedly laser excitation is assessed.As shown in Figure 7, the photoelectric current that is produced weakens along with the increase that exposes number of times really.Yet the amplitude that photoelectric current reduces is but less relatively.

Containing NADP ⁺Solution and the condition of 1:2 individual layer under, the generation at 340nm peak confirms to have formed NADPH in system of the present invention, and (Fig. 8 is [68-70] a).Under the electrochemical reduction condition, form NADP usually ⁺Dimer, these dimers also have absworption peak [68-71] at 340nm.

The schematic diagram of the photoelectric current production process of inferring of one aspect of the invention as shown in Figure 9.(Figure 10 may be that electronics is transferred to covalently bound fluorescein from gold surface by the double helix of DNA a) to the first step, thereby fluorescein is reduced into free radical anion.Described fluorescein free radical anion demonstrates has the long especially life-span (with hour calculating).Based on this reason, FI ^-Can the sufficiently long time of survival absorb photon.In case described fluorescein free radical anion has absorbed the fluorescein free radical anion (Figure 10 c) that the photon (Figure 10 b) of suitable energy has formed excitation state, it subsequently can be by the NADP to diffusion ⁺Electronics is provided and returns ground state.Yet, NADP ⁺Be two electron acceptors.Therefore, adjacent or even may be that identical chain is reduced once more and excites, for described NADP provides second electronics.In being included in system in the aqueous medium, this can promote NADP ^-Protonated.

Equation 1 relates to the measurement as the quantum efficiency of the feature of Optical Electro-Chemistry process.Quantum efficiency (Φ) may be defined as the electron number (dN that participates in described Optical Electro-Chemistry reaction _E-/ dt, electronics/second) with the number of photons (dN of time per unit photolytic activity molecule absorption _Hv/ dt, photons/second) ratio [7,8,12-14,16,21,24,30,32,33,36,37,40,72-76].

Adopting power is 4mW/cm2, and when the laser of λ=473 (5) nm excited, under the applying electrical potential of-750mV (vs.Ag/AgCl), the microelectrode of FI-DNA mark can obtain 450nA.cm ^-2Density of photocurrent.Identical with in solution the time (ε 473,43000M at the molar absorption coefficient of electrode surface to suppose FI-DNA ^-1Cm ^-1); Then the calculated value of described quantum efficiency is 0.25 (5).This numerical value is far longer than those to porphyrin SAM (0.1%) [35], the numerical value of the system that contains the multilayer pyrene (1%) [7] on gold surface report, and can with C ₆₀Numerical value in the SAM system [8,18,21-25] is compared.

Although disclose multiple embodiments of the present invention at this, those skilled in the art still can carry out all different transforms and modification within the scope of the present invention according to techniques well known.These modifications comprise with essentially identical method and reach identical purpose, the replacement of any aspect of the present invention being carried out with known equivalent.Number range comprises the numerical value that is used to limit this scope.Term used herein " comprises (comprising) " and is open term, be equivalent to term basically and " comprise (including), but be not limited to ", and term " contains (comprises) " and also has corresponding implication.Unless stated otherwise, singulative used herein " a ", " an " and " the " have also comprised plural situation.Therefore, for example, " object (a thing) " can comprise above a this object.This paper admits not that to quoting of list of references these lists of references are prior aries of the present invention.The priority document of quoting in this specification and all public publications, include but not limited to patent and patent application, be incorporated herein by reference in full at this, its effectiveness is equivalent to each publication separately and clearly state and be introduced into as a reference, and just as disclosing its full content in this article.The present invention also comprises whole embodiments and the variation that reaches substantially as previously mentioned with reference to described embodiment and accompanying drawing.

Embodiment 2

Material and preparation

Electrode: prepared golden microelectrode (50 μ m diameter) as previously mentioned and it is characterized [104].The gold net purchase is from Alfa Aesar (99.9% purity, 52 nets that are made into by the filament of 0.1mm diameter), and point is welded into Au (ibid) lead of 0.1mm diameter.By it is immersed the Piranha solution (H of boiling ₂O ₂: H ₂SO ₄=1: came in 10 minutes 3) the Au screen component is washed (operation Piranha etchant solution should be extremely careful, and it can not be stored in the closed container, and it is very strong oxidant, can with most of organic material vigorous reactions).

Fluorescein-DNA construct: (Saskatoon, SK Canada) adopt the synthetic and purify DNA of standard DNA synthetic method in the National Research Council.The sequence that is used for described photoelectric current experiment is as shown in table 3.Described base sequence is selected, minimizing optional secondary structure or tertiary structure, and introduced every kind of base of equivalent.

Table 3 is used for the dna sequence dna of photoelectric current research.The FI=fluorescein

1	HO-(CH 2) 6-S-S-(CH 2) 6-5’-GTCACGATGGCCCAGTAGTT-3’-FI
		2	5’-AACTACTGGGCCATCGTGAC-3’

The preparation of the gold electrode of FI-DNA modification: as previously mentioned, described microelectrode and net electrode are being contained the 50mM Tris-ClO of the fluorescein-labeled double-stranded DNA of 0.05mM ₄Hatch in the cushioning liquid (pH8.6) 5 days [104].

The photoelectric current condition: as shown in Figure 1, then with the described Tris-ClO of described electrode ₄Cushioning liquid cleans, and in equipment light inlet-electrochemical cell.If can be about to NAD (P) ⁺Adding to final concentration is 2mM.In order to get rid of the counterelectrode reaction that may influence chronoamperometry, must isolate described counterelectrode.With laser power is 4mW cm ^-2, wavelength is 473 ± 5nm and beam diameter (Intelite Inc.Genoa, NV is USA) as excitaton source less than the BM73-4V laser module of 0.8mm.Photoelectric current experiment is to carry out under employing is connected to the voltage clamp condition of Axopatch 200B amplifier (Axon Instruments) of CV 203BU headstage.The configuration of two electrodes is used for the voltage clamp condition, and reference electrode is the Ag/AgCl silk in the 1M KCl solution, and working electrode is the Au microelectrode of modification.Described spectroelectrochemistry battery is packed in the faraday's cage (Warner Instruments) of ground connection, and is positioned on activation air vibration proof (Kinetic System) platform.

Electric current passes through the filtering of low pass Bezier at 1kHz, and carries out digitlization at 5kHz by DigiData1322A (Axon Instruments), and by PClamp9.0 (Axon Instruments) record by PC control.Need further filtering, and adopt the low pass filter of 20Hz to realize by software approach.Analysis to all data is to use Origin7.0 (OriginLab Corporation) to carry out.Other electrochemical measurement is to use the potentiostat of the microelectrode customization of disposing for employing standard 3 electrodes to carry out.Described golden microelectrode (50 μ m diameter) can be used as working electrode.Contain 3M KCl and add in the glass tube that is stamped the Vycor tip by the Ag/AgCl silk is encapsulated into, made up reference electrode.By containing described electrolytical Luggin capillary described reference electrode is isolated from described battery.Described counterelectrode is a platinum filament.Before measuring, whole electrolyte solutions must be purified (purge) at least 20 minutes in Ar, and in measuring process, on described solution, keep being covered with Ar atmosphere.All experiments are all at room temperature carried out.

Result and discussion

Shift by electronics, infer to have formed stable chromophoric free radical anion.Can excite described chromophore by radiation (473nm) then.In this way, can suppress the inversion electron transmission.For example, fluorescein (FI) can be elected to be described chromophore, and use under suitable condition, have big absorptivity (ε 473=43000M thereby it is formed down in suitable reduction potential condition (750mV vs.Ag/AgCl) ^-1Cm ^-1) stable free radical anion.As shown in figure 13, in this embodiment, described chromophore is connected to described gold electrode by the double-stranded DNA of 20 base-pairs by the mercaptan key.Described DNA interval group can help prevent FI quencher owing to too close described electrode surface of excitation state, and the characteristic of semiconductor of described DNA also can assist electronics to transfer to described chromophore from described electrode simultaneously.Shown in the EPR spectroscopic methodology (Fig. 4), at applying electrical potential be-situation of 750mV (vs.Ag/AgCl) under, FI can form described radical anion FI ^-The electron acceptor that can select to be fit to promotes continuous electric current to flow.In exemplary, select NAD (P) ⁺(be NAD ⁺Or NADP ⁺) as electron acceptor, its reduction potential is higher than the reduction potential of described chromophore fluorescein.

Shown in Figure 14 a, at 473nm with 4mWcm ^-2Laser described microelectrode carried out radiation produced the electric current that continues, repeatedly amplitude only has a small reduction during radiation.Lacking NAD (P) ⁺Situation under, shown in Figure 14 b, described electric current has descended at least 50%.Shown in Figure 14 c, use red laser (632nm, the 10mW/cm of the nonabsorbable wavelength of fluorescein ²), do not observe electric current.Use does not have the individual layer of the fluorescein DNA of mark can not produce photoelectric current.NAD ⁺And NADP ⁺Produced the photocurrent quantum productive rate that equates.Shown in Figure 15 a, there is or lacking NAD (P) ⁺Situation under, between the intensity of described photon stream and electric current output, have linear relationship.Shown in Figure 15 b, described electric current reaches maintenance level at reduction potential during for-750mV, proves that FI was reduced into its free radical anion earlier before radiation, carries out the electronics transmission then.

Under the condition that FI excites, at applying electrical potential be-situation of 750mV (vs.Ag/AgCl) under, the microelectrode of FI-DNA mark has obtained 450nAcm ^-2Density of photocurrent.Suppose that FI-DNA is identical with in solution the time at the molar absorption coefficient of electrode surface; Then the calculated value of described efficient is 4 (1) photon-electrons ^-1(being equivalent to about 25% quantum yield).For the generation of NAD (P) H is described, implemented large-scale embodiment with golden net electrode, adopt AAS that described solution is monitored.Shown in Figure 16 a, there is the UV-vis peak of 340nm, it is feature (the ε 340=6220M that NADH shows when being subjected to radiation ^-1Cm ^-1).Verified, nicotinamide coenzyme can be formed the dimer of inanimate object activity by the free radical that the single electron reduction produces.In order to prove that NADH has BA and not to be dimer, in described solution, add alcohol dehydrogenase and acetaldehyde.Shown in Figure 16 b, the peak of 340nm disappears, and proves that the NADH that Optical Electro-Chemistry generates can carry out the conversion of zymetology application driving acetaldehyde to ethanol.According to estimation, the NAD of the abiology activity that has the 340nm peak equally that is generated ⁺Reduzate should be less than 1%.

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Claims (15)

1. Photocurrent generation system, including: (a) an electron transfer portion connected to the electrode through a conductive spacer portion; and (b) an electron accepting portion capable of accepting electrons from an excited electron transfer substance to form a reduced electron acceptor, wherein the electrode is biased to reduce the electron transfer moiety to form a reduced electron transfer species capable of absorbing photons to form the excited electron transfer species. 2. The system of claim 1, wherein the electron accepting moiety is located in an electron transfer solution. 3. The system of claim 2, wherein the electron transfer solution is an aqueous solution capable of donating protons to the reduced electron acceptor. 4. The system of claim 2, wherein the subsequent electron transfer moiety is immersed in the electron transfer solution for supplying the electron transfer material in the solution between the excited electron transfer species and the subsequent electron accepting moiety. repeated electron transfer reactions. 5. The system of claim 1, wherein the bias voltage applied to the electrode to form the reduced electron transfer species is lower than the voltage required to form the reduced electron acceptor. 6. The system of claim 1, wherein the rate of generation of the reduced electron transfer species is greater than the rate at which the excited electron transfer species donates electrons to the electron acceptor. 7. The system of claim 1, wherein the electron transfer moiety is fluorescein. 8. The system of claim 1, wherein the electrodes are gold electrodes. 9. The system of claim 1, wherein the conductive spacer moiety is a nucleic acid. 10. The system of claim 1, wherein the electron receiving moiety is NAD ⁺ or NADP ⁺ . 11. The system of claim 2, wherein the electron receiving moiety is NAD ⁺ or NADP ⁺ . 12. The system of claim 11, wherein an enzyme is further included in the electron transfer solution, and the enzyme utilizes NADH or NADPH as a cofactor. 13. The system of claim 12, wherein the enzyme is a dehydrogenase. 14. The system of claim 12, wherein the enzyme is alcohol dehydrogenase. 15. The system of claim 12, wherein the enzyme is a reductase.

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