The present application relates to methods and apparatus for performing an electrochemical activity at a localized region of an electrode surface. This has potential applications to electrodes, electrode arrays, devices, detectors for detecting various chemical events and so forth.

Background

A central tenet of electrochemistry is that any electrode must be connected to an external circuit via a wire such that a potential can be applied to the electrode or the electrode potential can be monitored. Implicit in this central tenet is that for multiple electrodes that operate independently in an electrode array, there must be a wire connecting each element of the array. There are two limitations to this requirement. Firstly, the connecting wires and associated bonding pads, plus the required gaps between electrodes to eliminate crosstalk, use considerable space on a chip surface and hence high density electrode arrays are difficult to achieve. The second consequence is that the position of each microfabricated conductive feature in an array must be preorganized. That is, the geometry of the array is intrinsically restricted to a choice made by the experimentalist prior to the experiment. The first of these limitations has independently been addressed both by Kelley et al. (L.

Soleymani, Z. C. Fang, E. H. Sargent and S. O. Kelley, Nat Nanotechnol, 2009, 4, 844-848.) and Matsue et al. (Z. Lin, Y. Takahashi, Y. Kitagawa, T. Umemura, H. Shiku, T. Matsue, Anal Chem 2008, 80, 6830-6833.) by reducing the number of connections required. In both cases this is achieved by forming columns and rows of electrodes in a grid pattern such that electrochemistry is confined to where the columns of electrodes cross over the electrode rows. The number of connections required is decreased because connectors are shared by many of these cross-over points.

It would be ideal if all connectors could be removed such that any location on an electrode surface can be switched on or off electrochemically. In this way much higher density of electrodes could be located on a surface and where the electrodes would not have to be preorganised. The concept of light-addressable potentiometric sensors (LAPS) to some degree fulfils these criteria. In LAPS, a silicon electrode is in physical contact with an electrolyte but electrically insulated from it by a silicon dioxide or silicon nitride coating. The LAPS device is illuminated with a light pulse at a discrete location and by measuring a surface photovoltage, the LAPS device gives information on local pH values or local redox potentials (G. Massobrio, S. Martinoia and M. Grattarola, Sensors Actuators B, 1992, 7, 484-487; G. X. Xu, X. S. Ye, L. F. Qin, Y. Xu, Y. Li, R. Li and P. Wang, Biosensors Bioelectronics, 2005, 20, 1757-1763.; T. Yoshinobu, H. Iwasaki, Y. Ui, K. Furuichi, Y. Ermolenko, Y. Mourzina, T. Wagner, N. Nather and M. J. Schoning, Methods, 2005, 37, 94-102.). However, the presence of the insulating layer between the silicon electrode and the solution in LAPS, means this technique is not able to drive electrochemical reactions. That is, Faradaic electrochemistry cannot be achieved using this technique.

It is an object of the present application to develop methods that enable

Faradaic electrochemistry to be performed on selected areas of the surface of an electrode. It is another object of embodiments of the present application to provide methods for performing Faradaic electrochemistry in a manner that leads to new apparatus and applications that address the problems associated with existing electrode arrays which have pre-determined architecture.

Summary of the Invention

For any semiconductor/electrolyte system, an applied potential exists at which the 10-1000 nm layer of the semiconductor, adjacent to the electrolyte, is depleted of charge carriers (X. G. Zhang, Electrochemistry of Silicon and its Oxide, Kluwer/Plenum, New York, 2001 .). This region in contact with the electrolyte, known as the space-charge layer (SCL), is of high-resistance while the bulk of the doped semiconductor remains of low electrical resistance. For instance, n-type semiconductors are depleted at potentials anodic of the flat- band potential, EFB (the potential where the charge carriers are the same at the electrolyte interface and the bulk). In the dark these depleted electrodes have insufficient charge carriers available to transfer charge, and charge transfer is kinetically limited. When the depleted sample is irradiated with light of adequate energy, a large number of charge carriers are generated. Electrons are excited to the conduction band with an equal number of holes in the valence band. This phenomenon is used in LAPS but cannot be used for processes where charge transfer occurs because of the insulating oxide layer between the

semiconductor surface and the electrolyte solution.

Using the example of silicon as the semiconductor material, the applicant has addressed this issue. The applicant realized that removal of the oxide layer would allow light to be used to facilitate charge transfer. However, it was recognized that upon applying a positive potential to the electrode, the surface layer of silicon would oxidise and insulate the electrode again. The applicant therefore developed a strategy to enable removal of the oxide layer, and to modify the surface in a manner that prevents surface re-oxidation. The work of the applicant commenced with the removal of the oxide layer by washing the semiconductor surface (in the test work, a silicon surface) in hydrofluoric acid or ammonium fluoride to give a hydrogen terminated silicon surface. The applicant then developed surface chemistry to stabilise the hydrogen terminated semiconductor (silicon) surface from oxidation. This is achieved using hydrosilylation chemistry, where the hydrogen terminated silicon surface is reacted with a molecule bearing a terminal alkene or alkyne such that a silicon- carbon covalent bond is formed. Surprisingly, if the hydrogen terminated silicon surface is modified in the hydrosilylation reaction with an organic molecule, for example a molecule that possesses an alkyne moiety at each end, then it was shown that such surfaces protect the silicon against oxidation so effectively that silicon electrodes can be used in aqueous solution for hundreds of redox cycles without oxidation of the underlying silicon.

This surface chemistry then led to the development of methods in which light could be used to allow Faradaic electrochemistry to proceed at a

semiconductor electrode, a concept referred to as light activated

electrochemistry, and the use of the technique to perform activities and processes not capable of being performed using existing technology. According to the present invention, there is provided a method for performing an electrochemical activity at a localized region of an electrode surface, the method comprising:

applying light to illuminate a region of a surface of an electrode, the electrode comprising:

a semiconductor substrate containing covalently bound organic molecules across the semiconductor substrate surface, and an electrochemically active species that is bound to the organic molecules or becomes bound to the organic molecules when a triggering event occurs,

wherein the applied light activates the illuminated region of the electrode surface to enable electrochemical activity to occur in that region, through the generation of charge carriers within the semiconductor substrate, which in turn drives an electrochemical activity at the electrochemically active species, with the non-illuminated regions of the electrode surface remaining insulating.

The electrochemical activity is typically Faradaic electrochemistry. The electrochemical activity may be an electrochemical reaction, such as a redox reaction. In some embodiments, the Faradaic electrochemistry occurs at the electrochemically active species (for example, where the electrochemically active species is a redox species). In other embodiments, the faradaic electrochemistry occurs adjacent the electrochemically active species (for example, where the electrochemically active species is a nanoparticle). In this second case, the electrochemically active species mediates a charge transfer between a redox species adjacent the electrochemically active species and the semiconductor substrate.

The applicant has identified that in order to achieve light activated

electrochemistry (i.e. an electrochemical activity at a given location on the surface of an electrode), certain conditions must be fulfilled. These conditions are satisfied by the method outlined above, and are as follows: 1 ) The oxide layer must be removed (this comes about through the requirement that the semiconductor substrate contains covalently bound organic molecules across the semiconductor substrate surface);

2) The semiconductor material or substrate, such as silicon, must be

protected against oxidation prior to, and during electrochemistry, using a protective organic molecule layer, such as nonadiyne;

3) An electrochemically active species that can collect electrons and donate electrons, such as a redox active species or a nanoparticle species, needs to be attached to the protective organic molecule layer (noting that the attachment only needs to be present when the electrochemical activity is taking place, and could be unattached otherwise); and

4) At the potential where electrochemical activity is observed, the

semiconductor substrate needs to generate charge carriers when illuminated, and to be depleted of charge carriers when not illuminated (so as to remain insulating). This can be achieved through matching the redox potential of the electrochemically active species with the semiconductor material (for example, matching to the silicon doping type in a silicon semiconductor) such that at the potential where Faradaic electrochemistry is observed, the silicon is in depletion when not illuminated.

If these criteria are fulfilled then light activated electrochemistry can occur anywhere on a monolithic semiconductor electrode surface with high spatial resolution such that high density electrode arrays can be made. The

electrochemistry can proceed to the surface bound electrochemically active species only or the electrochemically active species can then mediate charge transfer to a species in solution. As an example, the species in solution may be a redox species.

So what is described herein is a method using light to allow electrochemistry to occur anywhere on a semiconductor surface of an electrode with a micrometer scale spatial resolution, with the electrode requiring only a single electrical connection at its periphery. Light can switch on electrochemistry on the illuminated region, such that amperometry and voltammetry can be performed, while the rest of the surface not illuminated is an insulator.

The organic molecules are covalently immobilized across the semiconductor surface. This was achieved in the examples using the covalent immobilization of well-defined alkyne-terminated organic monolayers prepared on hydrogen terminated silicon in a single step hydrosilylation reaction with 1 ,8-nonadiyne. The step of covalently binding organic molecules across the semiconductor surface is key to protecting the silicon from oxidation such that Faradaic electrochemistry can proceed at the silicon surface. Subsequently, an electrochemically active species, or a redox species was attached to the distal end of the organic molecule. In the case of the examples, redox species used included azidomethylferrocene or 2-(azidomethyl)anthracene-9,10-dione, and these species were attached to the distal end of organic molecule (the alkyne) by copper(i) catalysed azide-alkyne cycloaddition reaction (click reaction). The examples demonstrate the methods for forming the basic layers, showing that Faradaic electrochemistry is switched in the presence of light, the spatial resolution for both bottom-side and topside illumination and the application of the technology for DNA arrays and the electrodeposition of soft conductive polymers, a viable approach for mask-free 2D "writing" of conductive features on surface. This technology is also able to provide high density electrode arrays that do not require predetermined electrode architecture.

The method described above may be used in a wide range of applications, and may be incorporated into many types of apparatus. As examples, the method is suitable for use in sensors, electrode sensors, electrode sensing arrays, electrochemical DNA sensor arrays, for forming components of molecular electronic devices (i.e. in forming connections to molecular electronic devices), for screening electrocatalysts, for use in instruments that combine

electrochemistry with fluorescence microscopy such as in cell biology, for use in devices that capture cells and release single cells for further study, for photovoltaic applications, in circuits, transistors or diodes, or for use in photoelectronic switches. In the case of sensors (i.e. sensors that detect the occurrence of an event, which is associated with an electrochemical activity), these may be incorporated into any type of apparatus that is for detecting the occurrence of that activity or event.

According to one aspect, there is provided an apparatus for detecting the occurrence of an electrochemical activity or a triggering event, the apparatus comprising:

(i) an electrode comprising a semiconductor substrate containing covalently bound organic molecules across the surface of the semiconductor substrate,

(ii) a light source for illuminating a region of the surface of the semiconductor substrate,

(iii) a voltage source for applying a potential to the electrode, and

(iv) a detector for detecting a change of electrical property indicative of the occurrence of an electrochemical activity or a triggering event.

According to another aspect, there is provided a sensor, electrode sensor, electrode sensing array, electrochemical DNA sensor array, molecular electronic device, screening apparatus, instrument, cell capture/release device, photovoltaic device, circuit, transistor, diode or photoelectronic switch, comprising an electrode, the electrode comprising:

a semiconductor substrate containing covalently bound organic molecules across the semiconductor substrate surface, and an electrochemically active species that is bound to the organic molecules or becomes bound to the organic molecules when a triggering event occurs,

wherein the semiconductor substrate material is matched to the redox potential of the electrochemically active species, such that when light is applied to a region of the electrode surface, light activates the illuminated region of the electrode surface to enable electrochemical activity to occur in that region, through the generation of charge carriers within the semiconductor substrate, which in turn drives an electrochemical activity at the electrochemically active species, with non-illuminated regions of the electrode surface remaining insulating. Brief Description of the Figures

Figure 1 presents a schematic illustration of the wet surface chemistry methods used in the hydrosilylation of 1 ,8-nonadiyne to develop a self-assembled monolayer (SAM) on Si(100), (20), according to one embodiment. The Si(100) wafer (20), comprising a native SiO _x layer (10) is first treated to remove the native SiO _x layer (10).

Figure 2 presents XPS spectra of monolayers assembled from 1 ,8-nonadiyne on a hydrogen-terminated Si(100) sample, (a) Survey spectrum, (b) Narrow scan of the C 1 s region, (c) High-resolution scan for the Si 2p region. Absent from the spectra is the signal associated with SiO _x species (102-104 eV). (d) Narrow scan of the 0 1 s region. Figure 3 presents XPS spectra of azidomethylferrocene-clicked nonadiyne modified Si(100) sample, (a) Survey spectrum. The narrow scan of the (b) N 1 s region, (c) Fe 2p, (d) C 1 s, (e) O 1 s region, and (f) Cu 2p. (g) High-resolution scan for the Si 2p region. Absent from the spectra is the signal associated with SiOx species (102-104 eV). (h) Narrow scan of the F 1 s.

Figure 4 presents cyclic voltammograms relating to the photooxidation of ferrocene functionalized n-Si(100). The illumination refers to the irradiation from a green light emitting diode (30 mA, 3.2 V, 1 .77 mW) from top with a

wavelength of 527 nm. The penetration depth (1/e) of the laser at 527 nm is about 1 .2 μηπ. The cyclic voltammograms were performed in the presence of aqueous electrolyte (1 .0 M HCIO ₄ ). The voltametric response for illuminated Si is indicated by (solid line) and (dashed line) in the dark.

Figure 5 presents (a) XPS spectra and high-resolution (b) Si 2p, (c) C 1s, and (d) N 1 s narrow spectrographs of AQ patterning silicon surface. Figure 6 presents cyclic voltammograms for anthraquinone functionalized poorly doped p-type Si (100) in B&R buffer (pH=10.06) in dark (dashed line) or under illumination (solid line). Figure 7 presents the results of a study showing the spatial resolution achievable using the technique of embodiments of the invention. The figure demonstrates schematic illustrations of the redox process (Figure 7(a)), the apparatus set-up (Figure 7(b)) showing a single peripheral lead (30), travelling optics (x-direction) (40), with a plot of catalytic current verses light pointer position (50) above the apparatus, and a series of graphs (Figure 7(c)) plotting the x-direction distance (μηπ) against the catalytic current (1/nA) for features of a given size (indicated above each graph). The actual feature sizes range from 300 μηι to 15 μηπ, with a Si thickness of 50-65 μηι and the spot size of light is 80 μηπ, 0.1 mW (laser operated at a constant current of 40 mA) continuous lasers light, Si is biased at a voltage of 0.2 V.

Figure 8 presents in a different format the light-addressable read-outs of the features of a size ranging from 300 to 50 μηι (as per Figure 7(c)), with a Si thickness of ~ 200 μηι and the spot size of light is 80 μηπ, 0.1 mW continuous laser light, Si is biased at a voltage of 0.2 V.

Figure 9 presents a graph of current signals versus distance of AQ

(anthraquinone) patterned silicon in Britton and Robinson buffer (pH=7.03). Figure 10 presents a schematic illustration of the principle of modified SECM (scanning electrochemical microscope) with light set-up (60) , in which a sample electrode is illuminated (~ 80 μηπ, 642 nm pulsed laser (62), 1 1 mW) from the back (bottom side illumination) to produce localized redox active process.

Figure 1 1 presents the results of surface generation-tip collection (SG/TC) mapping of redox events occurring on an electrode which is an azide-bearing, ferrocene-coupled, nonadiyne-modified n-type Si(100) electrode. The surface is free from any kind of pattern and has a single ohmic contact. The sample can be tilted up to 1 1 ± 7 μηι (Θ = 0.25 °). The current is represented by the grayscale colour as a function of x-y position. The colour is most intense at the peak illustrated in the figure, and the colour intensity fades to grey towards the periphery of the graph. The concentration of K ₄ [Fe(CN) ₆ ] in 0.1 M KNO3 is 10 mM, while the thickness of Si is 65 - 80 μηι.

Figure 12 presents cyclic voltammograms performed on a gold nanoparticle modified silicon surface passivated with a nonadiyne layer and

azidopropylamine, 'clicked' to the 1 ,8-nonadiyne via a Huisgen 1 ,3- cycloaddition at a scan rate of 100 mV s ^"1 in the presence of an aqueous solution of 1 mM Ru(NH ₃ ) ₆ Cl3 in 100 mM KCI. Faradaic oxidation and reduction of ruthenium hexamine was observed by shining light on the silicon (solid line) whereas no Faradaic response was observed when the silicon was in the dark (dashed line).

Figure 13 is a schematic illustration showing the fabrication of the DNA selective interface where probe DNA is attached to the silicon surface via the 'tandem click' reaction (step 1 ). Thereafter target DNA is hybridization onto the surface in a solution that contains hybridization buffer (step 2), followed by incubation in AQMS for 3 h followed by electrochemical measurement in an AQMS free solution (step 3).

Figure 14 presents schematic figures (Figure 14(a) and 14 (b)) and test results relating to the light activated electrochemical technique that allows electron transfer under illumination through DNA (Figure 14 (c)). A plot of photocathodic current (95) corresponding to the schematic of Figure 14(a) is displayed above the schematic shown in Figure 14(a). Figure 14(b) The probe-DNA (97) on Si is exposed to target DNA (99) being the noncomplementary (103), C-A mismatch (102), and complementary targets (101 ) and incubated in AQMS. The array of different DNA targets modified regions on the Si electrode were scanned from backside by a light pointer (100 mW). The plot (Figure 14 (c)) is of the x- direction distance (μηπ) along the electrode surface, plotted against the current readout showing complementary target (1 12), CA mismatch target (114), noncomplementary target (1 16) and then complementary target (112) again.

Figure 15 is a schematic illustration of the surface chemistry scheme for the capture and release of cells.

Figure 16 presents images of cell release using micromanipulation at the single cell level.

Figure 17 presents the results of a study on the stability of a ferrocene- functionalized electrode surface upon exposure and removal of light in 200 second cycles. The figure demonstrates the surface bound ferrocene-mediated charge transfer from an illuminated n-Si(100) electrode to an aqueous solution of 1 m M Fe(CN) ₆ ^4" in 0.1 M KN0 ₃ (0.1 M KN0 ₃ buffered at pH 7; substrate: 8- 12 Ω cm; illumination: 527 nm, ~1 .77 mW cm ^-2 ) and the inset shows the response of nonadiyne modified Si. The illumination refers to 527 nm

wavelength green light from top and the Si is biased at - 0.1 V while cycling the electrode.

Figure 18 is a schematic illustration of polypyrrole deposition onto non- structured, SAM modified photo-anode, n-type Si (all non-oxide semiconductors are prone to anodic decomposition). The flat band potential of n-type Si is {E _FB = - 0.34 V) and the oxidation of the monomer occurs at 0.2 V (E > E _F B). The spot size of the laser is about 80 μηι and the penetration depth (1/e) of the laser at 642 nm is about 3.3 μηι.

Figure 19 presents a FTIR Microscopy image of a deposited polypyrrole array on non-structured nonadiyne modified with the deposition time 2 s. The Si was biased at 0.24 V vs. Ag|AgCI during illumination from bottom with a distance of 500 μηι away from each other.

Figure 20 presents images and test results relating to light addressable drawing of a dot and lines using the technique of an embodiment of the invention; in which (a) a dot was made through light application for 1s, (b) line was made using 1000 μηπ/ο, (c) line was made using 2000 μηπ/ο and (d) a line was made using 3000 μηπ/α

Figure 21 is an image of a cartoon face made through light addressable drawing in accordance with an embodiment.

Figure 22 presents superimposed cyclic voltammograms showing the results of cyclic voltammetry of AQ terminated p-type Si (100) under different light intensities in B&R buffer (pH 10.09). The arrow indicates light intensity. The inserted graph shows the evolution of cathodic peak potentials (Epc) with light intensities.

Figure 23 presents images of (a) cobalt deposition under microscope; and (b) a scanning electron microscope (SEM) image showing the characteristic morphology of deposited cobalt.

Detailed Description of the Invention

Described herein is a method involving the use of light to allow electrochemistry to occur anywhere on a semiconductor surface of an electrode with a micrometer scale spatial resolution, with the electrode requiring only a single electrical connection at its periphery. Light can switch on electrochemistry on the illuminated region, such that amperometry and voltammetry can be performed, while the rest of the surface not illuminated is an insulator.

In the method of the present application, any portion of the electrode surface can be alternately illuminated to switch on the electrochemical activity, or darkened to switch off the electrochemical activity. Thus, the method may comprise the steps of: removing the light from the illuminated region to turn off the electrochemical activity.

The method may further comprise: illuminating a previously non-illuminated region of the electrode surface, to turn on the electrochemical activity. Electrode Arrays

As explained previously, this technology enables the provision of a high density electrode array that does not have any predetermined architecture. In contrast, in the prior art, spot welding of electrode terminals at individual electrode element locations on a device was required. Thus, in the method and any apparatus, the electrode suitably comprises a single connecting wire between the electrode and an external circuit.

In effect, the electrode may be considered to comprise multiple individual electrode elements which are connected to a single wire via the semiconductor substrate. The term "electrode element" is used to distinguish each location on the device surface which may be controlled independently, or operate as an electrode element separately from another location. In effect, each covalently bound organic molecule can be considered to constitute an individual electrode element. Each covalently bound organic molecule forms an electrode element that can be switched on when illuminated. Expressed another way, the electrode may be in the form of an electrode array, in which each covalently bound organic molecule constitutes an individual electrode element within the electrode array.

The electrode used in the method may be considered to be unstructured. In other words, the electrode is not pre-structured with electrical contacts at locations across a face of the electrode. This distinguishes the electrode from many forms of electrode arrays known in the art.

Additional features of the method

In a typical procedure for performing the method of the present application, a solution is brought into contact with the electrode. The solution is typically an electrolyte solution. Any suitable electrolyte may be used.

It is noted that the electrochemically active species may, in the course of the electrochemical activity or reaction, be itself modified (for example, it may be oxidized or reduced), or in the alternative, the electrochemically active species may mediate a charge transfer to a species in the solution. Where it mediates a charge transfer to a species in solution it may remain unreacted or unaffected by the electrochemical activity.

The method will typically involve a step of bringing an electrolyte into contact with the electrode and a second electrode. The electrolyte will typically be brought into contact with the electrode, the second electrode and a third electrode. The main electrode may be referred to as a working electrode or a light activated electrode, the second electrode may be referred to as a counter electrode, and the third electrode referred to as a reference electrode. The electrodes and electrolyte will typically form part of an electrical circuit that includes a voltage source.

The redox potential of the electrochemically active species is matched to the semiconductor composition such that at the potential where a redox reaction can occur, the semiconductor is depleted of charge carriers when not illuminated (darkened). Following from this, the method typically comprises a step of applying a bias potential to the electrode so that the charge carriers in non-illuminated regions of the electrode are in depletion, and charge carriers in illuminated regions of the electrode are generated and driven to the surface to become available for electrochemical activity. The type of reaction that can occur may be a reduction or oxidation. In some embodiments, the bias potential applied to the electrode is below the reduction potential of the electrochemically active species, so that when light is applied, the input potential exceeds the redox potential for the electrochemically active species, enabling a redox reaction to occur. In other embodiments, the bias potential applied to the electrode is above the oxidation potential of the electrochemically active species, so that when light is applied, the input potential exceeds the redox potential for the electrochemically active species, enabling a redox reaction to occur. The applied light generates an input potential that exceeds a threshold above which charge carriers are generated. Spatial resolution

As noted previously, and as demonstrated in the Examples below, this technology gives high spatial resolution between illuminated and insulating regions. The spatial resolution between an illuminated region enabling electrochemical activity to occur, and a zone where the semiconductor substrate surface remains insulating, can be 40 μηι or less, for example 35μηι or less, or even 30 μηι or less. This is demonstrated in the Examples.

Expressed another way, in the method, the application of light to said illuminated region of the electrode surface enables electrochemical activity to occur in that illuminated region, and to be prevented on the surface of the electrode a distance which is 100 μηπ, preferably 90, 80 μηπ, 70 μηπ, 60 μηπ, 50 μηπ, 40 μηπ, or most preferably 30 μηπ, away from the illuminated region.

The illumination may be front side illumination or back side illumination. Both topside and bottom side illumination have their advantages. With bottom side illumination the light source is not interfered with by any absorbing or scattering species between the light source and the surface. In contrast, with topside illumination the main advantage is high spatial resolutions obtained as there is less lateral diffusion of charged carriers at the silicon-solution interface. With bottom side illumination, the spatial resolution is dependent on the thickness of the silicon wafer and higher spatial resolution can be achieved with thinner silicon wafers and bottom-side illumination.

Unlike prior art devices, light dictates the electrode architecture. This enables very flexible performance of methods and for flexible devices to be developed, in which light is used to control the location of a reaction or similar.

When the method of the invention uses front side illumination, the temporal resolution of the light-activated chemistry may be 0.2 s, preferably 0.1 or most preferably 0.05 s.

When the method of the invention uses back side illumination, the temporal resolution of the light-activated chemistry may be 0.4 s, preferably 0.2 s or most preferably 0.1 s.

Following from this, in some embodiments, the application of light to the illuminated region of the electrode surface causes the electrochemical activity to occur at the electrochemically active species within 0.5 seconds, preferably within 0.2 seconds, or within 0.1 seconds. Electrochemical activity can even be activated or caused within 0.05 seconds of light application.

In some embodiments, the method comprises removing light from the illuminated region to turn off the electrochemical activity. Preferably, removal of light from the illuminated region turns off the electrochemical activity within 1 second, preferably within 0.8 seconds, 0.6 seconds, 0.5 seconds, 0.4 seconds, 0.3 seconds or 0.2 seconds.

In some embodiments, the method comprises alternately illuminating and darkening the electrode surface to switch electrochemical activity on or off, respectively. Preferably, illumination causes electrochemical activity to be switched on (at the location of illumination) within 0.5 seconds, 0.4 seconds, 0.3 seconds, 0.2 seconds or 0.1 seconds. Preferably, darkening causes

electrochemical activity (at the location or portion of the electrochemical surface that is darkened) to be switched off within 1 second, more preferably within 0.8 seconds, 0.6 seconds, 0.5 seconds, 0.4 seconds, 0.3 seconds or within 0.2 seconds.

Application of the method to detection processes

The method of the present application can be used to detect the occurrence of an electrochemical activity or a triggering event. In such applications, the method comprises detecting a change in an electrical property indicative of the occurrence of the electrochemical activity or triggering event.

The examples demonstrate a number of examples where the method is used in detecting electrochemical reactions (redox reactions) or associated triggering events. In such applications, the electrical property being detected can be a current, voltage or resistance, and usually a current.

The method for detecting the occurrence of an electrochemical activity or a triggering event, may further comprise monitoring an electrical signal output, and detecting for a signal that is associated with the occurrence of the electrochemical activity or triggering event.

Again, the electrical property or signal is suitably a current. The type of electrochemical activity may be a redox reaction. In the process, the

electrochemically active species may be oxidized or reduced.

One particular application of interest is the use of the method to detect the occurrence of DNA hybridization, so that the method can be used for detecting the presence of a particular target nucleic acid molecule in a sample. This has application to the detection of any suitable nucleic acid-containing species, including genetic material, microorganisms, bacteria, fungi, viruses, and so forth.

In such embodiments, the electrochemically active species may be unbound in the absence of a triggering event, and becomes bound on the occurrence of a triggering event, and the method comprises detecting for a change of electrical property indicative of the occurrence of the triggering event. The method may further comprise monitoring an electrical property output, to enable the detection of the change in the electrical property output. The electrical property output may be described as an electrical signal output.

When applied to these applications, in the light application step, light is applied to the electrode surface enables electrochemical activity to occur, to thereby drive a redox reaction at illuminated organic molecule locations where the electrochemically active species has become bound, indicating the occurrence of the triggering event. Further, as foreshadowed above, the triggering event may be DNA

hybridization. In this instance:

- the organic molecules covalently bound to the semiconductor substrate surface are coupled to a probe nucleic acid molecule having a nucleotide sequence that is at least substantially complementary to the nucleic acid sequence of a target nucleic acid molecule, and

the method comprises:

- bringing a sample to be tested for the presence of the target nucleic acid molecule into contact with the semiconductor substrate surface,

- bringing a solution comprising an electrochemically active species capable of co-ordinating with any hybridized pair of probe and target nucleic acid molecules into contact with the semiconductor substrate surface, resulting in binding of the electrochemically active species to the organic molecule via the hybridized pair of probe and target nucleic acid molecules,

- applying light to the electrode surface to drive a redox reaction at locations where hybridization and electrochemically active species binding has occurred, and

- detecting a change in an electrical property indicative of the occurrence of hybridization.

Background information on hybridization technology

Hybridization occurs when hydrogen bonds form between complementary nucleotide bases, for example, T-A, C-G, and A-U. Complementary nucleic acids comprise complementary bases with the capacity for precise pairing between two nucleotides, for example, if a nucleotide at a certain position in the sequence of nucleotides of an single-stranded nucleic acid (the target nucleic acid molecule) is capable of hydrogen bonding with a nucleotide at the same position in the sequence of nucleotides of a probe nucleic acid molecule, then the single-stranded nucleic acid target and probe are considered to be complementary to each other at that position. The single-stranded nucleic acid and the probe are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Accordingly, complementary does not necessarily mean that two hybridizing nucleic acid strands have 100% nucleotide complementarity in the hybridizing region. For example, in some embodiments, hybridizing nucleic acids can have less than 100%

complementarity, less than 95% complementarity, less than 90%

complementarity, less than 85% complementarity, less than 80%

complementarity, in the hybridizing region provided that the complementarity is sufficient to promote hybridization under the conditions used. In preferred embodiments, however, the hybridization occurs between specific

complementary sequences and not between non-complementary sequences. Indeed, as explained in further detail below, the present application allows for detection of the presence of a nucleic acid sequence that is perfectly

complementary to the probe nucleic acid sequence, to the exclusion of other nucleic acid sequences having one non-complementary base in the sequence.

In some embodiments of the methods and apparatus described herein, a target nucleic acid molecule can contain at least one nucleic acid sequence that can hybridize to the nucleic acid sequence of the probe. Such sequences that can hybridize include complementary nucleotides. In certain embodiments, a sequence that can hybridize can contain a contiguous sequence of

complementary nucleotides. For example, a nucleic acid sequence can contain at least one contiguous sequence section complementary to at least one sequence in capture probe. In such embodiments, the at least one contiguous sequence of complementary nucleotides contained in the probe nucleic acid sequence can have a length of at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 25 nucleotides, at least 30 nucleotides, or at least 40 nucleotides, as examples.

The number of non-complementary nucleotides in the sequences of the target and probe is preferably not more than 5, not more than 4, not more than 3, not more than 2, not more than 1 , and preferably there are no non-complementary nucleotides. As is known in the art, the ability of a target single-stranded nucleic acid molecule and probe nucleic acid molecule to hybridize to one another can be modulated by varying the conditions in which the hybridization occurs. Such conditions are well known in the art and can include, for example, pH, temperature, concentration of salts, and the presence of particular molecules in the hybridization reaction. Under conditions of low stringency, a probe nucleic acid molecule and a single-stranded nucleic acid with a low degree of complementarity (therefore not the target nucleic acid sequence) may be able to hybridize to one another. Conversely, under more highly stringent conditions, only probes and target single-stranded nucleic acids with a high degree of complementarity are likely to hybridize to one another. The present application allows the detection of exact complementary target nucleic acid sequences, and the separate detection of a second nucleic acid molecule that is not the target, and contains non-complementary nucleotides in the sequence compared to the target. This provides a powerful technique for precise detection and analysis.

Additional details of methods for detecting target nucleic acid molecules

Following from the discussion of hybridization above, according to some embodiments, the probe nucleic acid molecule contains a nucleic acid sequence that is perfectly complementary to the nucleic acid sequence of a target nucleic acid molecule.

The method will typically involve the step of bringing the sample to be tested for the presence of the target nucleic acid molecule into contact with the

semiconductor substrate surface to result in hybridization between the probe and target nucleic acid molecules.

According to preferred embodiments, the detection step comprises detecting for a change in the electrical property of a magnitude that indicates the occurrence of hybridization of perfectly complementary nucleic acid sequences of the target and probe nucleic acid molecules, the magnitude of the change being different to that obtained from hybridization of a non-perfectly complementary nucleic acid sequence in the sample to the nucleic acid sequence of the probe nucleic acid molecule.

The change in electrical property to be detected is typically a change in

Faradaic current signal. Consequently, the detection step may comprise detecting for a change in current of a magnitude that indicates the occurrence of hybridization of perfectly complementary nucleic acid sequences of the target and probe nucleic acid molecules, and wherein the current change is of a different magnitude to the current change detected when a non-perfectly complementary nucleic acid sequence in the sample is hybridized with the nucleic acid sequence of the probe nucleic acid molecule.

In applications of the method to DNA hybridization processes, the

electrochemically active species is suitably a redox active intercalator. The types of electrochemically active species (or redox molecules, particularly organic or organometallic redox molecules) described below may be used in this regard. Such species may be modified, for example by sulphonic acid modification, to improve solubility in solution.

The organic molecule covalently bonded to the semiconductor substrate surface may be coupled to the probe nucleic acid molecule via a linker. Linkers are described in further detail below in the discussion of the chemical components.

Definitions relevant to hybridization applications

The term "nucleic acid" refers to a nucleotide polymer, and unless otherwise limited, includes analogs of natural nucleotides that can function in a similar manner (e.g., hybridize) to naturally occurring nucleotides. Unless otherwise limited "nucleic acids" can include, in addition to the standard bases adenine, cytosine, guanine, thymine and uracil, various naturally occurring and synthetic bases (e.g., inosine), nucleotides and/or backbones.

The term nucleic acid includes any form of DNA or RNA, including, for example, genomic DNA; complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification; and mRNA.

The term nucleic acid encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e, a double-stranded nucleic acid need not be double-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modification thereof, such as by methylation and/or by capping. Nucleic acid modifications can include addition of chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and functionality to the individual nucleic acid bases or to the nucleic acid as a whole. Such modifications may include base modifications such as 2-position sugar modifications, 5-position pyrimidine modifications, 8-position purine

modifications, modifications at cytosine exocyclic amines, substitutions of 5- bromo-uracil, backbone modifications, unusual base pairing combinations such as the isobases isocytidine and isoguanidine, and the like.

More particularly, in certain embodiments, nucleic acids, can include

polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of nucleic acid that is an N- or C- glycoside of a purine or pyrimidine base, as well as other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. The term nucleic acid also encompasses linked nucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499, 6,670,461 , 6,262,490, and 6,770,748, each of which is incorporated herein by reference. The nucleic acid(s) (particularly those of the probe nucleic acid sequence) can be derived from a completely chemical synthesis process, such as a solid phase-mediated chemical synthesis, from a biological source, such as through isolation from any species that produces nucleic acid, or from processes that involve the manipulation of nucleic acids by molecular biology tools, such as DNA replication, PCR amplification, reverse transcription, or from a combination of those processes.

The term "target nucleic acids" is used herein to refer to particular nucleic acids to be detected in the method of embodiments of the present application.

Cell tethering and cell release applications

The method of the present application may alternatively be used to perform target tethering or target release on the application of light to a region of the electrode surface.

According to such embodiments, the method is for performing target tethering or target release, wherein the electrochemically active species has an oxidized state and a reduced state with one state tethering the target and the other state releasing the target, and the method comprises:

- applying light to a region of the electrode surface to cause a redox reaction involving the electrochemically active species, to thereby cause target tethering or target release from the surface of the electrode.

The target may be of any suitable type capable of being tethered through the electrochemically active species. In some embodiments, the target is a cell.

The electrochemically active species may be in either an oxidized or reduced state when the target is in a tethered position, and a redox reaction may be driven through light application to cause cell release. In some embodiments, the target is tethered when the electrochemically active species is in the oxidized state and released when the electrochemically active species is in the reduced state. In other embodiments, the target is tethered when the electrochemically active species is in the reduced state and is released when the

electrochemically active species is in the oxidized state.

In some embodiments, the target is tethered to the electrode surface via the organic molecule prior to the light application step, and the method comprises applying light to a region of the electrode surface to cause reduction of the electrochemically active species, resulting in cleavage of a tethering bond retaining the target and causing release of the target from the surface of the electrode. In an alternative embodiment, the opposite reaction is used. That is, the target may be tethered to the electrode surface via the organic molecule prior to the light application step, and the method comprises applying light to a region of the electrode surface to cause oxidation of the electrochemically active species, resulting in cleavage of a tethering bond retaining the target and causing release of the target from the surface of the electrode.

Any suitable chemistry for the electrochemically active species may be selected that enables target tethering and release on oxidation (or in the converse case, reduction) of the electrochemically active species. In one embodiment developed by the applicant, the electrochemically active species contains a ring closing group that closes on reduction of the electrochemically active species with the cleavage of a bond that tethers the target, and the method comprises applying light to cause reduction of the electrochemically active species, resulting in ring closure of the ring closing group and release of the target from the surface of the electrode. In one example, the electrochemically active species comprises a hydroquinone in the oxidized state and a lactone in the reduced state.

Other chemistry can be used to cause release of the target on oxidation of the electrochemically active species.

In the case of target cell release, in some embodiments it is desirable to release single cells. For example, the ability to release single cells is important for the study of cell heterogeneity and for further analysis of single cells with aberrant behavior. In the case of single cell release, the spatial resolution that can be achieved is important. Top side illumination has been found to be useful in this case, since top side illumination provides improved spatial resolution to provide single cell release capabilities.

Polymer or metal electrodeposition

The method of the present application may alternatively be used to perform electrochemical activity to electrodeposit a polymer or metal onto a localized region of the electrode surface. According to this process, the electrochemically active species is bound to the organic molecules, the electrode surface is contacted with an electrolyte comprising monomer or metal cations, and the method comprises applying light to a region of the electrode surface to drive the electrodeposition of the polymer or metal onto the illuminated region of the electrode surface.

A key advantage of this application for the process relates to the simple way in which the location of polymer/metal deposition is controlled. As an example, the polymer or metal can be deposited onto the semiconductor substrate surface by a drawing process in which light is drawn along a pathway across the semiconductor substrate surface to deposit the polymer or metal on the substrate surface along the drawn pathway. In an alternative, light can be patterned onto the surface through a mask, template, laser source, or otherwise.

One example of this is the electrodepositing of a polymer onto the

semiconductor surface. The polymer may be conductive or non-conductive. Examples of suitable polymers are outlined further below. Another example is electrodeposition of metal onto a semiconductor surface. Any suitable metal may be deposited in this way, and examples are outlined further below. Chemistry

Semiconductor substrate

Any suitable semiconductor materials may be used in the method and apparatus of the present application. Such semiconductors are well known in the art. The semiconductor substrate may, for example, be selected from the group consisting of intrinsic, n-type, p-type, multilayer, silicon, titanium dioxide, zinc sulphide, gallium arsenide, indium tin oxide, gallium selenide, indium phosphide, tungsten selenide, boron arsenide, bismuth vanadate and cadmium selenide semiconductor substrates.

In some embodiments, the semiconductor substrate is a silicon semiconductor substrate. For example the semiconductor substrate may be a p-type silicon semiconductor substrate, such as a low doped, p-type silicon semiconductor substrate. One particular example is p-type Si(100). Alternatively, the semiconductor substrate may be n-type silicon semiconductor substrate, such as a low doped, n-type silicon semiconductor substrate. An example is n-type Si(100).

According to particular embodiments, the semiconductor substrate is a doped silicon semiconductor substrate, and the redox potential of the

electrochemically active species is matched to the doping type such that at the potential where a redox reaction can occur, the semiconductor is depleted of charge carriers when not illuminated.

As explained at the outset, the semiconductor substrate surface is oxide free. This is achieved through the covalent bonding of an organic species, or organic molecules, across the semiconductor surface. The organic molecules form an organic monolayer across the semiconductor substrate surface. In particular, the organic molecules form a self assembled monolayer across the

semiconductor substrate surface.

The organic molecules form an organic monolayer across the semiconductor substrate surface that is sufficiently thin to allow electron transfer between the semiconductor substrate and any bound electrochemically active species, in the presence of light, while preventing oxidation of the semiconductor surface. The organic molecules comprise a headgroup attached to the silicon surface, and a tail. The headgroup may be an atom such as a carbon atom or a nitrogen atom, and is typically carbon. In the case of a carbon headgroup atom, the carbon may be an alkyl, alkenyl or alkynyl carbon atom. The tail is typically an organic tail, such as an aliphatic hydrocarbon tail. Aromatic groups may also be present in the organic tail.

The organic molecules covalently bonded to the semiconductor substrate surface may be described as organic surface passivating molecules. They provide surface passivation of the semiconductor surface, such as the silicon surface. Surface passivation relates to the protection of the surface from oxidation.

Any type of organic molecule that provides surface passivation effect, while allowing charge or electron transfer to the end of the group, may be used.

Examples may be selected from the group consisting of tethered alkyl diyne groups, tethered azidoalkyl thiol groups, alkenes, carboxylic acid-substituted alkenes, alkene such as undecylenic acid, undecylene succinimidyl ester, 1 ,4- diethynylbenzene, 1 -heptyne, 1 -hexadecene, 1 1 -chloro-1 -undecene, 1 1 -bromo- 1 -undecene, 2-bromo-ethyl ester, 1 -octene, methyl terminated silicon by either alkyl Grignard or an alkyl lithium reagent, 1 -pentene, 1 -octyne, phenyl, aryl diazonium salts, alkanethiols, 10-undecynoic fluoride.

When covalently bonding the suitable organic molecule to the semiconductor surface, there may be modification of the group to form the tethering bond to the surface, such as the silicon surface. Thus, where an alkyl diyne is used, the alkyl diyne is reacted with the surface semiconductor surface atom (such as silicon), to convert one alkyne bond to an alkene bond. Thus, the "tethered alkyl diyne group" comprises an alkene group (or headgroup) attached to the semiconductor surface. Despite this conversion, the organic molecule may be referred to interchangeably as either an alkyl diyne, a tethered alkyl diyne, or similar.

According to some examples, the organic molecules are tethered C6-C30 alkyi diyne groups, such as tethered C6-C18 alkyi diyne groups or tethered C6-C15 alkyi diyne groups. Tethered C9 alkyi diyne is used in the examples, but groups of differing alkyi chain length may be used.

The electrochemically active species may be bound directly to the organic molecule, or it may be bound to the organic molecules via a linking group.

Any group may be used that links the organic molecule to the electrochemically active species. As examples, the linking group may be selected from the group consisting of tethered diazides, tethered azide-terminated thiols, tethered azide- terminated lactones, amines, carboxylic acids, thiols to maleimides, nitrile, nitrous oxide, diazoalkanes, azomethine, azoxy-compounds, carbonyl oxides, nitroso-oxides, carbonyl imines, ozone, acetals, vinylcarbenes, vinylazenes, ketocarbenes, iminoazenes, iminocarbenes, pyrrolines, pyrrolidines, pyrazoles, pyrazzolines, pyrazolidines, imidazolidines, oxazoles, oxazolines, aziridines, oxazolidines, triazolines, trizolidines, tetrazoles, pentazoles, aryldiaziridine, hydroxysuccinimide esters, sulfonamides , thiophenes, epoxides,

semicarbazides, azidobutanes, aryl azides, carbodiimodes, N,N'-disuccinimidyl carbonates, acridinium esters, heterobifunctional aliphatic crosslinkers, PEG based crosslinkers, multifunctional scaffolds, homobifunctional crosslinkers, biotinylation reagents, reactive fluorescent dyes, reactive phycobiliproteins, reactive tandem dyes, bifunctional lanthanide chelators, and modified or activated oligo probes.

The electrochemically active species may be any species capable of collecting electrons and/or donating electrons. The electrochemically active species may for example be a redox species. The electrochemically active species may therefore be selected from the group consisting of organic/organometallic redox molecules and nanoparticles. As explained above, the electrochemically active species is bound to, or may become bound to, the organic molecule (which is itself covalently bonded to the semiconductor substrate surface). Any chemistry that allows binding between the organic molecule and the electrochemically active species may be used. As an example, the organic molecule may initially contain an alkyne bond at one end, and the electrochemically active species may contain an azide group, so that the two can be coupled through click chemistry. Click chemistry is well known in the field of organic chemistry, and relates to azide-alkyne

cycloaddition. This is typically catalyzed by a catalyst, such as a copper (I) catalyst. Following from this, in some embodiments, the electrochemically active species may be attached to the organic molecule through an azide- alkyne cycloaddition reaction. As examples, the electrochemically active species may be an azide-substituted redox species or a 1 ,2,3-triazole- substituted redox species. The 1 ,2,3-triazole is the group that results following attachment of the electrochemically active species to the organic molecule.

In cases where the electrochemically active species is attached to the organic molecule via a linking group, then the click chemistry may be used at one or both ends of the linking group, to attach one end of the linking group to the organic molecule, and/or to attach the electrochemically active species to the other end of the linking group. Thus, in such embodiments, electrochemically active species may be attached to the organic molecule via a linking group, where the attachment between either the organic molecule or the

electrochemically active species and the linking group is through an azide- alkyne cycloaddition reaction.

As examples of suitable organic/organometallic redox molecules, there may be mentioned hydroquinones, anthroquinones, metal-sandwich complexes, metallocenes, bis(aromatic) metal complexes, organic dyes, lactones, redox- active ring opening/closing compounds, benzoquinones, anthraquinones (including disulfonic acid anthraquinone or sulfonic acid anthraquinone), napthoquinones such as 1 ,2-napthoquinone-4-sulfonic acid, porphyrins such as 21 H,23H-porphine-2,18-dipropanoic acid, phenazines such as Benzo[a]phenazine-5-sulfonic acid, phthalocyanines such as copper

phthalocyanine 3, 4', 4", 4"'-tetrasulfonic acid, acridines such as L-glutaminyl-L- glutaminyl-L-seryl-L-isoleucyl-L-a-glutamyl-L-glutaminyl-L-l eucyl-L-a-glutamyl- N1 -[3-[(6-chloro-2-methoxy-9-acridinyl)amino]propyl] ^" (9CI), phenothiazines such as phenothiazine-2-acetic acid, quinolones such as oxolinic acid,

anthraquinones such as doxorubicin, metal complexes containing intercalating ligands such as 9,10-diimine phenathrenequinone or chrysene, porphyrins such as copper(ll) tetrakis(4-N-methylpyridyl)porphyrin, phthalocyanines such as cuprolinic blue, acridines such as lucigenin), phenothiazines such as methylene blue, phenazines such as safranine T and phenanthridines such as ethidium.

The organic/organometallic redox molecule may be an azide-substituted redox molecule or a 1 ,2,3-triazole-substituted redox molecule, which is suitable for connection to alkyne terminated organic molecules (or linking groups) through the azide-alkyne cycloaddition as mentioned above. Accordingly, it follows that the organic/organometallic redox molecule may be selected from the group consisting of: an alkylazide substituted hydroquinone, an alkylazide substituted anthraquinone, an alkylazide substituted metal-sandwich complex, an alkylazide substituted metallocenes, bis(aromatic) metal complexes, an alkylazide substituted dye, an alkylazide substituted lactone, an alkylazide substituted redox-active ring opening/closing compound, an alkylazide substituted benzoqinone, a 1 ,2,3-triazole-substituted hydroquinone, a 1 ,2,3- triazole-substituted anthraquinone, a 1 ,2,3-triazole-substituted metal-sandwich complex, a 1 ,2,3-triazole-substituted metallocenes, a 1 ,2,3-triazole-substituted bis(aromatic) metal complexes, a 1 ,2,3-triazole-substituted dye, a 1 ,2,3-triazole- substituted lactone, a 1 ,2,3-triazole-substituted redox-active ring

opening/closing compound, a 1 ,2,3-triazole-substituted benzoqinone, an alkylazide substituted hydroquinone, an alkylazide substituted anthraquinone, an alkylazide substituted napthoquinone, an alkylazide substituted, porphyrin, an alkylazide substituted phenazine, an alkylazide substituted phthalocyanine, an alkylazide substituted acridine, an alkylazide substituted phenothiazine, an alkylazide substituted quinolone, an alkylazide substituted anthraquinone, an alkylazide substituted metal complex containing an intercalating ligand, an alkylazide substituted porphyrin, an alkylazide substituted phthalocyanine, an alkylazide substituted acridine, an alkylazide substituted phenothiazine, an alkylazide substituted phenazine, an alkylazide substituted phenanthridine, a 1 ,2,3-triazole-substituted hydroquinone, a 1 ,2,3-triazole-substituted anthraquinone, a 1 ,2,3-triazole-substituted napthoquinone, a 1 ,2,3-triazole- substituted porphyrin, a 1 ,2,3-triazole-substituted phenazine, a 1 ,2,3-triazole- substituted phthalocyanine, a 1 ,2,3-triazole-substituted substituted acridine, a 1 ,2,3-triazole-substituted phenothiazine, a 1 ,2,3-triazole-substituted quinolone, a 1 ,2,3-triazole-substituted anthraquinone, a 1 ,2,3-triazole-substituted metal complex containing an intercalating ligand, a 1 ,2,3-triazole-substituted porphyrin, a 1 ,2,3-triazole-substituted phthalocyanine, a 1 ,2,3-triazole- substituted acridine, a 1 ,2,3-triazole-substituted phenothiazine, a 1 ,2,3-triazole- substituted phenazine and an alkylazide substituted phenanthridine.

In some embodiments, the electrochemically active species is a nanoparticle. The nanoparticles may be selected from gold nanoparticles, carbon

nanoparticles, carbon nanotubes, graphene sheets, nobel metal nanoparticles and semiconducting nanoparticles. Nanoparticles may be attached to each organic molecule, directly or through a linking group. In other embodiments nanoparticles are attached to at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the organic molecules across the semiconductor substrate surface.

Any chemistry may be used for the attachment of the nanoparticles known in the art. As one example, the nanoparticles may be attached via a thiol linking group.

Polymers and metals for deposition techniques

In embodiments where the method is for performing electrochemistry to electrodeposit a polymer onto a localized region of the electrode surface, any type of polymer that is formed through the oxidation of the corresponding monomer can be used. Such polymers may be conductive or non-conductive. This mechanism is common for the formation of conductive polymers, so this method has particular application to the electrodeposition of conductive polymers. In this case, the method may comprise the step of electrodepositing onto the semiconductor substrate surface a polymer from a class selected from the group consisting of: polyaniline, polypyrrole, polycarbazole, polyindole, polyazepine, polythiophene (including poly (3,4-ethylenedioxythiophene), polyphenylene, polyfluorene, polypyrene, polyazulene, polynaphthalene, polyacetylene, polyaromatics and polyheteroaromatics, poly(3,4- ethylenedioxythiophene) polystyrene sulfonate (PEDOT-PSS), poly(p- phenylene sulfide), poly(p-phenylene vinylene), poly(phenyleneethylene), polythienylenevinylene, polyisothianaphthene, polyfuran, polystyrene, polyisoprene, polybutadiene, poly(3-octylthiophnene-3-methylthiophene), poly(p-phenylene-terephthalamide), poly(oc-naphthylamine), polythiophene- vinylene, poly(2,5-thienylenevinylene), poly(isothianaphthene)and derivatives and co-polymers thereof, including co-polymers of any of the preceding classes with a polyvinyl polymer.

In embodiments where the method is for performing electrochemistry to electrodeposit a metal onto a localized region of the electrode surface, the method may comprise the step of electrodepositing onto the semiconductor substrate surface a metal selected from the group consisting of: copper, silver, gold, cobalt, platinum, nickel, palladium, lead, iron, zinc, ruthenium and rhodium.

Apparatus

In general terms, the present application provides an apparatus for detecting the occurrence of an electrochemical activity or a triggering event, the apparatus comprising:

(i) an electrode comprising a semiconductor substrate containing covalently bound organic molecules across the surface of the semiconductor substrate,

(ii) a light source for illuminating a region of the surface of the semiconductor substrate,

(iii) a voltage source for applying a potential to the electrode, and

(iv) a detector for detecting a change of electrical property indicative of the occurrence of an electrochemical activity or a triggering event.

The apparatus may be in the form of a sensor, electrode sensor, electrode sensing array, electrochemical DNA sensor array, molecular electronic device, screening apparatus, instrument, cell capture/release device, photovoltaic device, circuit, transistor, diode or photoelectronic switch.

It follows from this that the present application also provides a sensor, electrode sensor, electrode sensing array, electrochemical DNA sensor array, molecular electronic device, screening apparatus, instrument, cell capture/release device, photovoltaic device, circuit, transistor, diode or photoelectronic switch, comprising an electrode, the electrode comprising:

semiconductor substrate containing covalently bound organic molecules across the semiconductor substrate surface, and an electrochemically active species that is bound to the organic molecules or becomes bound to the organic molecules when a triggering event occurs,

wherein the semiconductor substrate material is matched to the redox potential of the electrochemically active species, such that when light is applied to a region of the electrode surface, light activates the illuminated region of the electrode surface to enable electrochemical activity to occur in that region, through the generation of charge carriers within the semiconductor substrate, which in turn drives an electrochemical activity at the electrochemically active species, with non-illuminated regions of the electrode surface remaining insulating.

The description provided above of preferred features or embodiments of the method that read onto physical features of the device apply equally to the apparatus and device.

In addition to those features, it will be understood that the apparatus may comprise, or may be separately coupled to, a light source which is used in the operation of the apparatus. The light source could be of any suitable type known in the art, including those that apply light of a specific wavelength, or broad spectrum light. The light source may comprise lenses and/or mirrors to focus the location of light application. The apparatus may further comprise a light source application controller for controlling the location of light application to the surface of the semiconductor substrate. This may be by way of a mechanical control system, or track system, for controlling light application, a series of mirrors or lenses for controlling the light application, or a combination of both. Any arrangement that may suit a particular application can be used. The light source application controller will typically be one that allows light to be applied to different regions of the semiconductor substrate surface at different times during the detection process.

The apparatus will also generally comprise an electrolyte receiving zone positioned to allow electrolyte to come into contact with the surface of the semiconductor substrate. This may be through the arrangement of the device in the form of a cell containing the electrode, a zone or passage or region for receiving electrolyte. This may be located within a casing that also positions a second electrode, or counter electrode, within the electrolyte receiving zone.

Particularly for embodiments where there is a flow of sample, or electrolyte through the apparatus, the apparatus may comprise an electrolyte reservoir which is in fluid communication with the electrolyte receiving zone.

The apparatus will also generally comprise a second electrode. This may be positioned to contact electrolyte when the electrolyte is located in the electrolyte receiving zone. The apparatus may also comprise a third electrode, also positioned to contact the electrolyte when electrolyte is located in the electrolyte receiving zone. The third electrode may be a reference electrode.

The voltage source of the apparatus is used for applying a bias potential to the electrode. Any voltage source can be used, such as a battery or a mains power supply.

The apparatus will typically also contain a controller for controlling operation of the apparatus. This may be in the form of a computer. There may also be a user interface for providing a signal to indicate the occurrence of an

electrochemical activity or triggering event. The signal may be a visual signal, such as a light or screen read-out, or may be a signal of another type, such as a sound. The signal may be an electronic signal that is transmitted to a separate device, where the occurrence of the electrochemical activity or triggering event is flagged, recorded, or otherwise. In some embodiments, the user interface is a visual interface for providing a visual signal to indicate the occurrence of an electrochemical activity or triggering event.

In embodiments where the apparatus is one for detecting the occurrence of a triggering event associated with a sample, and the apparatus comprises a sample inlet for receiving a sample.

One particular application is for detecting the presence of target nucleic acid molecule in a sample. In this instance, the organic molecules covalently bound to the semiconductor substrate surface are coupled to a probe nucleic acid molecule having a nucleotide sequence at least substantially complementary to the nucleic acid sequence of the target nucleic acid molecule, and the apparatus comprises:

- a sample inlet for receiving a sample,

- a flow system that enables a sample received in the sample inlet to come into contact with the semiconductor substrate surface, and enables a solution comprising an electrochemically active species capable of co-ordinating with any hybridized pair of probe and target nucleic acid molecules to come into contact with the semiconductor substrate surface, and

- an interface for providing a signal to indicate whether hybridization between the probe and target nucleic acid molecules has occurred.

In another application the apparatus is used to perform target tethering or target release, the apparatus comprising:

(i) an electrode comprising a semiconductor substrate containing covalently bound organic molecules across the surface of the semiconductor substrate, and an electrochemically active species bound to the organic molecules, the electrochemically active species having an oxidized state and a reduced state, with one state capable of tethering a target and the other state capable of releasing the tethered target,

(ii) a light source for illuminating a region of the surface of the semiconductor substrate, and

(iii) a voltage source for applying a potential to the electrode.

It is also noted that the apparatus in this embodiment may further comprise

(iv) a detector for detecting a change of electrical property indicative of the tethering or release of the target.

In terms of the types of targets to be tethered and released, and other relevant details, reference is made to the discussion above in the context of the corresponding method. The target may be a cell according to particular embodiments. The target may be initially tethered to the electrode surface via the organic molecule. Following application of the light source, the target is released.

It is possible for this technique and apparatus to therefore be used in situations where there are a range of cells across the electrode surface, and there is controlled release of selected cells, from the selected regions, through the application of light. This may find application in a range of micro-devices, such as in the case of molecular electronic devices, and in bionic applications.

The apparatus may comprise a light source application controller for controlling the location of light application to the surface of the electrode, to control the location to which the target can be tethered, or from which the target can be released. The light source application controller may allow light to be applied to different regions of the semiconductor substrate surface at different times during the operation of the apparatus.

For applications relating to target (such as target cell) tethering and release, the apparatus suitably comprises components that allow a target cell to be loaded onto the electrode surface, ready for performing a cell release operation. This may be achieved through the provision of various fluid passageways for loading. Thus, the apparatus may suitably comprise a fluid receiving region positioned to allow fluid to come into contact with the surface of the

semiconductor substrate. There is also suitably provided a fluid reservoir which is in fluid communication with the fluid receiving region. The apparatus then may suitably also contain a target loading inlet in fluid communication with the fluid receiving region, to allow loading of a target species into the apparatus. This fluid may be an electrolyte, or an electrolyte containing the target to be loaded onto the electrode surface. Control of the fluid flow may be by way of a flow system for controlling flow of fluid and target through inlets and into contact with the electrode. This may be operated by a computer. There may be any suitable combination of valves and passageways that provide the required function.

In general terms, the apparatus (or devices) will comprise a second electrode, or a counter electrode. The voltage source referred to previously is used to applying a bias potential to the electrode. This enables the operation of the apparatus in the manner described above. Again, the operation of this and the other components of the apparatus may be controlled by a controller. The controller may comprise a user interface for providing a signal to indicate the occurrence of the relevant event, such as target tethering or target release. The user interface may be visual interface as described previously.

Chemical Definitions

In the above, reference is made to a range of organic chemical components, and the following section may be used to understand the scope of those references.

The term "alkyl" used either alone or in a compound word denotes straight chain, branched or mono- or poly- cyclic alkyl, preferably C1 -30 alkyl or cycloalkyl, more preferably C1 -18 alkyl. The preferred alkyl chain length for alkyl-containing hydrocarbon tails for the organic molecules are C1 - C18, such as C2 - C18, C4 - C18 or C6-C15 alkyl groups. Such chain lengths may also suit the linking group, and any other alkyl group component or substituent.

Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, amyl, hexyl, heptyl, octyl, nonyl, decyl, 1 -, 2- , 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, undecyl, dodecyl and the like. Examples of cyclic alkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl and the like. The alkyl group may optionally contain one or more substituents, examples of which are indicated below.

The term "alkenyl" used either alone or in compound words such as

"alkenyloxy" denotes groups formed from straight chain, branched or cyclic alkenes including ethylenically mono-, di- or poly-unsaturated alkyl or cycloalkyl groups as defined above, preferably C2-30, more preferably C2-24 alkenyl. Examples of alkenyl include vinyl, allyl, 1 -methylvinyl, butenyl, iso-butenyl, 3- methyl-2-butenyl, 1 -pentenyl, cyclopentenyl, 1 -methyl-cyclopentenyl, 1 -hexenyl, 3-hexenyl, cyclohexenyl, 1 -heptenyl, 3-heptenyl, 1 -octenyl, cyclooctenyl, 1 - nonenyl, 2-nonenyl, 3-nonenyl, 1 -decenyl, 3-decenyl, 1 ,3-butadienyl, 1 - 4,pentadienyl, 1 ,3-cyclopentadienyl, 1 ,3-hexadienyl, 1 ,4-hexadienyl, 1 ,3- cyclohexadienyl, 1 ,4-cyclohexaidenyl, 1 ,3-cycloheptadienyl, 1 ,3,5- cycloheptatrienyl and 1 ,3,5,7-cyclooctatetraenyl. The term "alkenyl" also encompasses dienes, such as the 1 ,(X-1 )-C _x -dienes. In the case of X=9, this refers to a nonadiene, with the double bonds located at the C1 and C8 carbon atoms, which corresponds to 1 ,8-nonadiene. Thus, examples of dienes in this regard include 1 ,8-nonadiene, 1 ,9-decadiene. Alkenes containing an additional alkyne bond are also encompassed. The alkenyl or alkene may optionally contain one or more substituents, examples of which are indicated below.

The term "alkynyl" used either alone or in compound words such as

"alkynyloxy" denotes groups formed from straight chain, branched or cyclic alkynes, containing at least one alkyne triple bond, preferably C2-30, more preferably C2-24 alkynyl. The alkynyl may further comprise one or more double bonds, or any other suitable substituent(s). Examples of alkynyl include 1 - butynyl, 1 -pentynyl, 1 -hexynyl, 1 ,5-hexadiyne, 1 -heptynyl, 1-octynyl, 1 -nonynyl, 2-nonynyl, 8-nonynyl, 1 -decenyl, 1 ,5,8-nonatriynyl, and so forth. The term "alkynyl" also encompasses diynes, such as the 1 ,(X-1 )-C _x -diynes. In the case of X=9, this refers to a nonadiyne, with the double bonds located at the C1 and C8 carbon atoms, which corresponds to 1 ,8-nonadyene. Thus, examples of diynes in this regard include 1 ,8-nonadiene, 1 ,9-decadiene. With further coupling, the diyne may be converted into the corresponding diene. Alkynes containing an alkene bond are also encompassed. The alkynyl or alkyne may optionally contain one or more substituents, examples of which are indicated below.

The term "alkoxy" used either alone or in compound words such as "optionally substituted alkoxy" denotes straight chain or branched alkoxy, preferably C1 -30 alkoxy. Examples of alkoxy include methoxy, ethoxy, n-propyloxy, isopropyloxy and the different butoxy isomers.

The term "acyl" used either alone or in compound words such as "optionally substituted acyl" denotes carbamoyl, aliphatic acyl group and acyl group containing an aromatic ring, which is referred to as aromatic acyl or a

heterocyclic ring which is referred to as heterocyclic acyl, preferably C1 -30 acyl, or C1 -20 acyl, or C1 -C12 acyl. Examples of acyl include carbamoyl; straight chain or branched alkanoyl such as formyl, acetyl; alkoxycarbonyl such as methoxycarbonyl; cycloalkylcarbonyl such as cyclopropylcarbonyl; alkylsulfonyl such as methylsulfonyl; alkoxysulfonyl such as methoxysulfonyl; aroyl such as benzoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl,), naphthylalkanoyl (e.g. naphthylacetyl); aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl), naphthylalkenoyl (e.g. naphthylpropenoyl); and

heterocyclicalkanoyl such as thienylacetyl;.

The term "aryl" used either alone or in compound words such as "optionally substituted aryl" or "optionally substituted heteroaryl" denotes single,

polynuclear, conjugated and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring systems. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, pyridyl, 4-phenylpyridyl, 3- phenylpyridyl, thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazolyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. The aromatic heterocyclic ring system may contain 1 to 4 heteratoms

independently selected from N, O and S and containing up to 9 carbon atoms in the ring.

In this specification, any organic group, such as that of the organic molecule, the linking group, or the electrochemically active species (where this is organic or organometallic), may contain one or more substituents. The substituents may be selected from one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, aryloxy, benzyloxy, haloalkoxy, haloalkenyloxy, haloaryloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, amino, alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, benzylamino, dibenzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, diacylamino, acyloxy, alkylsulphonyloxy, arylsulphenyloxy, heterocyclyl, heterocycloxy, heterocyclamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, benzylthio, acylthio, phosphorus-containing groups and the like. The substituents should be non-deleterious substituents, which refers to any of the substituents outlined above which does not interfere with the operation of the electrode as a light addressable electrode.

Examples

The present application will now be described in further detail with reference to a number of examples, that demonstrate the formation of the electrode, its operation, and its utility in performing the range of methods described herein. Example 1 : Formation self-assembled monolayer (SAM) on silicon (Si)

To protect Si from oxidation, the SAM was formed on Si(100) (dopant

concentration, N _D , ~ 4.5 * 10 ¹⁴ cm ^-3 , resistivity 8-12 Ω cm) from 1 ,8-nonadiyne (S. Ciampi, T. Booking, K. A. Kilian, M. James, J. B. Harper, J. J. Gooding, Langmuir, 2007, 23, 9320-9329.). This is illustrated schematically in Figure 1 . Si(100) wafer (20), comprising a native SiO _x layer (10) is first treated to remove the native SiO _x layer (10). Si wafers were cut into pieces (~ 20 x 30 mm), rinsed with redistilled DCM and dried under a stream of argon. Si was immersed in hot piranha solution (1 vol. 30 % by mass aqueous hydrogen peroxide/3 vol.

sulphuric acid) for ~ 1 h. The temperature of the piranha was 100 °C. Samples were rinsed with copious amount of Milli-Q H ₂ 0. Then Si was transferred to a hydrofluoric acid (2.5 %) solution for 90 s to have a hydrogen terminated surface. The Si wafer was immediately transferred to a degassed (a minimum of five freeze-pump-thaw cycles) 1 ,8-nonadiyne. Extra care was always taken to exclude air completely from the custom-made Schlenk flask by using an argon stream. The reaction mixture was kept in an argon atmosphere and placed in an oil bath at 165 °C for 3 h. After cooling down to room temperature, the nonadiyne modified Si was rinsed with redistilled DCM several times. Then the modified Si placed in redistilled DCM in a test tube (air was excluded from the tube by a stream of an argon flow). The tube is wrapped with aluminium foil and put in the refrigerator (4 °C) for overnight.

XPS spectra were acquired on the nonadiyne modified n-type Si. See Figure 2. The survey scan indicates the presence of Si, C, and O, which correlates with the formation of an organic monolayer on the silicon substrate (G. F. Cerofolini, C. Galati, S. Reina, L. Renna, Appl. Phys. A 2004, 80, 161-166.; G. F. Cerofolini, C. Galati, S. Reina, L. Renna, Surf. Interface Anal. 2006, 38, 126- 138.; G. F. Cerofolini, C. Galati, S. Reina, L. Renna, Mater. Sci. Eng. C 2003, 23, 253-257.). The narrow scan of Si 2p shows no significant oxide or suboxide silicon in the range of 102-104 eV (F. J. Himpsel, F. R. McFeely, A. Taleb- Ibrahimi, J. A. Yarmoff, G. Hollinger, Phys. Rev. B 1988, 38, 6084-6096.; A. Lehner, G. Steinhoff, M. S. Brandt, M. Eickhoff, M. J. Stutzmann, Appl. Phys. 2003, 94, 2289-2294.; S. Ciampi, T. Booking, K. A. Kilian, M. James, J. B. Harper, J. J. Gooding, Langmuir, 2007, 23, 9320-9329.). The absence of any significant level of oxide indicates a well-defined monolayer which prevents the oxidation of the silicon substrate (A. B. Sieval, R. Linke, H. Zuilhof, E. J. R. Sudholter, Adv. Mater. 2000, 12, 1457-1460.; P. Gorostiza, C. H. De Villeneuve, Q. Y. Sun, F. Sanz, X. Wallart, R. Boukherroub, P. Allongue, J. Phys. Chem. B 2006, 110, 5576-5585.; S. Ciampi, P. K. Eggers, G. Le Saux, M. James, J. B. Harper, J. J. Gooding, Langmuir 2009, 25, 2530-2539.). The O 1s emission at ~ 532 eV is attributed to adsorbed oxygen.

The electrochemistry of the 1 ,8-nonadiyne modified silicon surface was then recorded in 1 mM Fe(CN) ₆ ^4_ in 0.1 M KN0 ₃ . In both the dark and the light no discernible Faradaic electrochemistry was observed demonstrating the necessity for a surface bound electrochemically active element.

Example 2: Ferrocene functionalized nonadiyne modified SAM on n-type Si(100)

To prepare a nonadiyne modified n-type Si(100) (resistivity 8-12 Ω cm, E _F B is - 0.28 ± 0.03 in 0.1 M KN0 ₃ ) with an electrochemically active element to be attached, 2-propanol, copper (II) sulphate (0.40 mM) and Milli-Q H ₂ 0 were degassed for ~ 20 mins. To a custom made reaction tube containing the nonadiyne modified n-type Si( 00): (i) azidomethylferrocene (8.67 mM in 2- propanol); (ii) copper(ll) sulphate pentahydrate (0.40 mM) and (iii) sodium ascorbate (20.19 mM in Milli-Q H ₂ 0) were added. Reactions were carried out at room temperature with the exclusion of air from the reaction environment by an argon flow and put in the dark for 45 mins. The azidomethylferrocene bound surface was then rinsed thoroughly with redistilled ethanol and Milli-Q H ₂ O. A final quick rinse of 0.5 M HCI was employed to remove any residual copper from the reaction, followed by MilliQ H ₂ O and redistilled ethanol.

XPS spectra of the resultant surface indicated the successful formation of ferrocene-derivatized Si(100) with in situ generation of catalytically active Cu(i) species. See Figure 3. The survey spectra showed nitrogen and iron-related emission peaks with Si, carbon, and oxygen signals. The detailed investigation of N 1 s, Fe 2p and C 1 s of the XPS spectra indicated the successful triazole formation. The narrow scan for Si 2p showed the absence of detectable emission related with silicon oxide species at (102-104) eV similar to the observation for nonadiyne modified surfaces and indicated high quality ferrocene derivatized surfaces (S. Ciampi, T. Booking, K. A. Kilian, M. James, J. B. Harper, J. J. Gooding, Langmuir, 2007, 23, 9320-9329.; A. B. Sieval, R. Linke, H. Zuilhof, E. J. R. Sudholter, Adv. Mater. 2000, 12, 1457-1460.). The coupling efficiency was found to be ~ 32 %.

To demonstrate the concept, the photoanodic properties of azidomethylferrocene functionalized n-type Si was studied using cyclic voltammetry (CV) in the presence of aqueous electrolyte of 1.0 M HCIO ₄ . See Figure 4. The voltametric response for illuminated Si is indicated by (solid line) and (dashed line) in the dark. It is evident from the CV that light can switch on electrochemistry on the illuminated region as the photogenerated minority carriers, holes are driven to surface and become available for redox reactions. In the dark, the poorly doped n-type Si is in depletion which causes kinetic limitation to charge transfer with the electrolytic solution and there is no Faradaic response in dark.

Example 3: Attachment of Anthraquinone onto alkyne terminated p-type Si (100)

Azide-terminated anthraquinone (AQ) was covalently attached to alkyne terminated p-type Si (100) (boron doping density 2.14 ^χ 10 ¹⁵ cm ^"3 , resistivity 10- 20 Ω cm, flatband potential -0.16 V vs. 3M Ag/AgCI) via a Cu (l)-catalyzed alkyne-azide cycloaddition reaction to yield AQ modified electrodes. Alkyne- functionalized silicon surface were placed in 2 ml_ dimethyl sulphoxide containing 2 mM of azide-anthraquinone, 9.76 mM of copper (I) bromide, and 20.6 mM tetramethylethylenediamine. Reactions were carried out at room temperature, in the dark, without excluding air from the reaction environment and stopped after 30 min by removal of the modified sample from the reaction vessel. The prepared surface-bound samples were rinsed consecutively with copious amounts of water, ethanol, and then the samples were rinsed with plenty of dichloromethane to remove the photoresist, blown dry under argon, and stored under argon before use.

XPS analysis was employed to support the formation of AQ modified silicon surface. See Figure 5. As expected, the presence of nitrogen species was evident after the 'click' reaction on the nonadiyne modified silicon surface. It is clearly shown on Si 2p narrow scans acquired from AQ patterned area that there is still no oxidation of the substrate. The high-resolution N1 s XPS spectra for micropatterned AQ are fitted to two distinct peaks having binding energies of 401 .7 and 400.3 eV and a 1 :2 ratio of the integrated area, which are corresponding to N-N=N and C-N-N, respectively. The presence of the triazole group indicated the success for alkyne-azide cycloaddition reaction of anthraquinone on the surface. And it is further verified by high-resolution C1 s XPS narrow scans in Figure 5. c. The low energy peak centered at 283.8 was attributed to C-Si, and C1 s peaks with binding energy at 285.0 and 284.5 eV were ascribed to carbon-bonded carbon in aliphatic and aromatic configuration, while two more functions centered at 288.0 eV (C=0 for carbonyl group) and 286.5 eV (C-N) are existing on silicon surface.

Cyclic voltammetry of the anthraquinoine modified p-type Si(100) surface was performed in the dark (dashed line in Figure 6) and the light (solid line in Figure 6). In the dark there was no evidence of Faradaic electrochemistry of the anthraquinone, while well-defined redox peaks were shown when silicon is under illumination indicating photo-generated electrons were capable of reducing the anthraquinone species on the silicon surface. Example 4: Spatial resolution of the light activated electrochemistry with bottom side illumination

Having shown that light can activate electrochemistry of the surface bound redox species, we also measured the feature size of the redox events. This is illustrated schematically and the data presented in Figure 7. Microfabrication was used to covalently attach ferrocene at precise regions and sizes on the monolithic nonadiyne-passivated n-type Si(100). The nonadiyne modified Si was spin-coated with SU-8 2007 photoresist (Microchem) at 3,000 rpm for 30 s. The sample is then baked on a hot plate at 65 °C for 2 min followed by 3 min at 95 °C. To form patterns the SU-8 photoresist was exposed to UV light for 22 s under a photomask with the desired pattern (Quintel Q6000 Mask Aligner) followed by repeating the baking process. The sample was then developed by immersing the wafer in SU-8 developer (Microchem) for 2 min, rinsed in 100% isopropyl alcohol and dried under nitrogen flow. The area of the SU-8 photoresist that was not exposed to UV light is thus removed, results in the formation photoresist patterns. Subsequently azidomethylferrocene was 'clicked' onto the exposed regions to determine the spatial resolution.

A focused light source is scanned from the backside, across the ferrocene region and the current recorded as a function of the travelling distance by the light source. The measured catalytic current from surface tethered ferrocene units gives an indication about spatial resolution achievable with light activated electrochemistry. The read-outs of the feature decrease when the thickness of Si thinned down to (50-65) μηπ. See Figure 8. This is due to the shorter diffusion length travelled by the carriers. The read-out for the lowest feature size of 15 μηι was found to be ~ 53 μηι (Figure 8). The thinner Si provides short diffusion length, hence the read-out decreased with the actual feature. On the contrary, the read-outs of actual feature size do not decrease even if we decrease the width of modified region from 300 μηι to 50 μηι for thicker Si (~ 200 μηπ). Example 5: Spatial and temporal resolution of the light activated

electrochemistry with top side illumination of anthraquinone on lightly doped p-type Si (100) surface

Anthraquinone was patterned onto a p-type Si(100) surface as described above for ferrocene. A laser diode was scanned horizontally across the AQ patterned surface such that the illuminated area became electrochemically active. The variation in feature size recorded electrochemically versus the actual size of the anthraquinone modified region showed, relative to bottom side illumination, the minimum feature size that was recorded was 33 μ(Ύΐ for a 15 μιη anthraquinone line. These results are presented in Figure 9.

The nonadiyne-modified poorly-doped, p-type silicon (100) wafer was modified with an azide-containing anthraquinone species using a Huisgen 1 ,3-dipolar cycloaddition reaction. -0.3 V vs. Ag/AgCI/0.1 M KCI was applied to the surface. A Faradaic signal was observed 0.05 s after the surface was illuminated from the top side and 0.2 s when the surface was illuminated from the back side whereas the surface was electrochemically inactive 0.1 s after the light was switched off from the top side and 0.2 s when from the back side. The results are summarized in Table 1 below.

Table 1 Electrochemical response times for top and back side illumination

Example 6: Demonstrating light activated electrochemistry can be used to detect redox active species in solution

To demonstrate that light activated electrochemistry can be used to monitor electrochemistry to species in solution, a scanning electrochemical microscope (SECM) was used in the surface generation-tip collection mode. See Figures 10 and 1 1 . The light set-up (60) includes directing light from a 642 nm pulsed laser (62) through a fiber collimation package (63) and subsequently a collimated laser (1 .3 mm FWHM output beam diameter) (65), the light then being focused using a plano-convex lens (66) (ca. 80 μηι FWHM focused spot size). As shown, a single peripheral lead (30) is used in the apparatus. A plot (70) of tip current versus x-y position is shown at the top of Figure 10. In surface generation-tip collection (SG/TC) mode, a reaction that occurs on the substrate is probed by the tip (Scanning Electrochemical Microscopy, Marcel Dekker, Inc., New York, USA, 2001 ). The n-type Si photoanode (80) surface has ferrocene clicked on the continuous non-structured nonadiyne monolayer without any kind of pattern while the detected species in solution was K4[Fe(CN) ₆ ). In SG/TC mode, the Pt tip was hold at 35 μηι away from the electrode and moved in the x- and y-direction in steps of 20 μηι to map the electrochemistry as the Pt tip crossed the illuminated region and the light activated redox event was recorded.

It was also observed that the detected feature size became smaller when the concentration of the redox couple in the solution increased. This may be due to the charge hopping between the adjacent ferrocene groups as charge hopping between surface bound ferrocene may become dominant at the lower concentration of the redox couple in the solution, results in the increase of the feature size. Example 7: Detecting redox species in solution using a nanoparticle electrochemically active element

In a first example, a nonadiyne modified n-type Si(100) surface is modified with a short azidomethyl thiol linker to which gold nanoparticles are attached. The surface is placed in a solution of 1 mM K ₃ [Fe(CN) ₆ ] in 50 mM KCI. In the dark no Faradaic electrochemistry is observed but in the light it is.

Colloidal gold nanoparticles with a size of 15-25 nm were prepared by adding 1 .75 ml_ of 1 % sodium citrate to 50 ml_ of boiling 0.01 % HAuCI ₄ .3H ₂ 0 with vigorous stirring. Boiling was done for 10 mins and cooled with vigorous stirring. The nonadiyne modified silicon wafer modified with azidomethyl thiol and incubated in gold suspension for about 6 h. After that, the wafer were rinsed with Milli-Q, ethanol, ethyl acetate, DCM and followed by drying under a stream of argon.

AFM images confirm the presence of the nanoparticles on the surface and although some clusters were observed on the surface, the nanoparticles were randomly dispersed across the surface.

The silicon surface as a working electrode, a gold wire as the counter electrode and Ag/AgCI/3 M NaCI as a reference electrode were used. The cyclic voltammograms were performed at scan rate of 20 mV s ^"1 in the presence of an aqueous solution of 1 mM K ₃ [Fe(CN) ₆ ] in 50 mM KCI. Faradaic oxidation and reduction of ferricyanide was observed by shinning the silicon with light, whereas in the dark there was no Faradaic response.

In a second example, a nonadiyne modified poorly doped p-type Si(100) surface is modified with a short azidopropylamine linker to which gold nanoparticles are attached. The surface is placed in a solution of 1 mMRu(NH ₃ ) ₆ Cl3 in 100 mM KCI. In the dark, no Faradaic electrochemistry is observed but in the light it is. Colloidal gold nanoparticles with a size of 15-25 nm were prepared by adding 1 .75 ml_ of 1 % sodium citrate to 50 ml_ of boiling 0.01 % HAuCI4.3H20 with vigorous stirring. Boiling was done for 10 mins and cooled with vigorous stirring. The nonadiyne modified silicon wafer was modified with azidopropylamine via a Huisgen 1 ,3-dipolar cycloaddition reaction and incubated in a gold nanoparticle suspension for 3 h. After that, the wafer was rinsed with Milli-Q water. SEM images confirm the presence of the nanoparticles randomly dispersed across the surface.

The silicon surface as a working electrode, a platinum mesh as the counter electrode and an Ag/AgCI/0.1 M KCI reference electrode was used. The cyclic voltammograms were performed at a scan rate of 100 mV s-1 in the presence of an aqueous solution of 1 mM Ru(NH ₃ ) ₆ CI ₃ in 100 mM KCI. Faradaic oxidation and reduction of ruthenium hexamine was observed by shining light on the silicon whereas no Faradaic response was observed when the silicon was in the dark. The Faradaic response is shown in Figure 12.

Example 8: Light activated electrochemical detection of DNA hybridization

The detection of DNA hybridization was performed amperometrically using long range charge transfer through the DNA to an intercalated anthraquinone monosulfonic acid (AQMS). This is illustrated schematically in Figure 13. In this case p-type Si(100) with (500 μηπ, 10-20 Ω cm) was used. Table 2 below shows the DNA sequences. DNA was attached to the surface using a click reaction of alkyne-DNA on the 1 ,8-nonadiyne modified Si(100) sample by adding 20 μΙ_ of: (a) degassed (~ 20 min) 1 ,3-diazidopropane (800 μΜ in 3:1 :2 DMSO/H ₂ 0/f-BuOH, degassed), (b) CuBr (490 μΜ) and Ν,Ν,Ν',Ν'- tetramethylethane-1 ,2-diamine (TMEDA, 1030 μΜ) in 3:1 DMSO/H ₂ 0, and (c) 200 μΜ alkyne-DNA (refer step 1 of Figure 13). The final concentration of alkyne DNA in reaction medium was 6.67 μΜ. Table 2: | DNA sequences

The click reaction was carried out for 1 h at room temperature and stopped by removal of the solution from the modified sample, followed by rinsing with EtOH, MilliQ, and EtOH again. Single-stranded surfaces were incubated for ~ 5 h in a solution containing 1 % bovine serum albumin (BSA) and 0.05% sodium dodecyl sulphate (SDS) in 0.05 M phosphate buffer (K ₂ HP04/KH ₂ P04) containing 0.13 M NaCI, pH 7.0. The reaction was stopped by washing with 0.05 M phosphate buffer in 0.13 M NaCI. Then DNA targets were added (20 μΜ, 60 μΙ_ in 0.05 M phosphate buffer containing 0.2 M NaCI, pH 7). The sample was put in the hybridization buffer for 2 h (refer step 2 of Figure 13). Hybridisation was stopped by rinsing the samples thoroughly with 0.05 M phosphate buffer pH 7 in 0.2 M NaCI and then incubated in 1 mM anthraquinone-2-sulphonic acid (AQMS) in 0.2 M KCI for ~ 3 h (refer step 3 of Figure 13). The hybridized ds-DNA will be intercalated with AQMS and will take part in light activated electrochemistry. In the absence of AQMS no electrochemistry was observed in the light or the dark, hence the presence of the DNA double strands formed during hybridization with the target strand allows the AQMS to become surface bound, as required for light activated electrochemistry, and the DNA duplexes could be detected.

An array with the same sequence of probe DNA attached at discrete locations on the electrode was formed but each location exposed to a different target sequence; complementary, mismatched sequence and no target DNA. See Figure 14. The light set-up (60) shown in Figure 14 (a) is identical to that shown in Figure 10 and includes directing light from a 642 nm pulsed laser (62) through a fiber collimation package (63) and subsequently a collimated laser (1 .3 mm FWHM output beam diameter) (65), the light then being focused using a plano-convex lens (66) (ca. 80 μηι FWHM focused spot size). As shown, a single peripheral lead (30) is used in the apparatus. AQMS within the oval shape (90) indicates a redox active intercalator anthraquinone -2-sulfonic acid. As shown in Figure 14 (b), individual elements were exposed to either complementary target (101 ), single base pair mismatch (102) or

noncomplementary (103) target sequences and incubated in AQMS as discussed above. A scanning light pointer 105 is shown in Figure 14 (b). A plot 108 above the schematic of Figure 14 (b) shows one wire read-outs in 2D. The entire surface was poised at -0.550 V as the AQMS had a reduction potential at around -500 mV versus Ag|AgCI| 3M NaCI and then the light source scanned across the different elements of the array from back. The plot (Figure 14 (c)) is of the x-direction distance (μηπ) along the electrode surface, plotted against the current readout. The reduction current was significantly greater for scanning the region exposed to complementary than for the other two sequences.

Example 9: Cell release at specific area

Light activated electrochemistry can also be used to selectively release things ("targets") from the surface. Poorly doped p-type Si (100), is modified with 1 ,8- nonadiyne and then an electrochemically-cleavable linker was attached on silicon surface. To this linker is attached an antibody to capture rare cells as shown in the Scheme in Figure 15. This is illustrated using the cancer cell line MCF-7 cells. By illuminating specific regions of the surface using a microscope, cell imaging and cell/s release at specific areas were carried out.

The process of silicon modification for cell release system consists of many steps. Firstly, an electrochemically-cleavable linker, azide-terminated lactone, will be attached to the end of the monolayer using a 'click' reaction with a copper catalyst (shown in step a) of Figure 15). Followed in step b) the chemical oxidation will cause ring opening of the as mentioned lactone into benzoquinone, leaving a carboxyl group at the surface. In step c) of Figure 15 the distal carboxyl group will then be activated by 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide) (EDC)/N-Hydroxysuccinimide (NHS) coupling and then an ethylene oxide (E0 ₆ ) will be attached. The terminal OH group is activated with a disuccinimidyl carbonate (DSC) linker to immobilize the anti-epithelial cell adhesion molecule antibody that binds to the EpCAM within the cells. Lastly, as seen in step d), a negative potential (E=-1 .2 V vs Ag/AgCI/3 M NaCI, for 5 min) will result in the ring-closure of the hydroquinone back to the lactone, and in doing so, releasing the cells from the surface.

The antibody modified silicon wafer was fixed in the Teflon chamber. The MCF- 7 cells (10 ⁵ cm ^"2 ) were incubated on the above surface for 15 minutes, and then stained with Hoechst 33342 (1 :500 in culture media) for 15 min.

Further, by reducing the illuminated spot size, a small area was under illumination. And then a negative potential of -1 .2 V vs. Ag/AgCI/3 M NaCI was applied on the surface for 5 min. Along with the switch reaction to the lactone, the cells under illumination, down to a single cell, were expected to be released and recovered through the use of micromanipulation for downstream analysis and viability assays. The process is illustrated in Figure 16. The results of this downstream analysis are summarised in Table 3 below.

Table 3 Electrochemical response times for top and back side illumination

In Figure 16, micromanipulation is shown to not be effective on the predetermined individual cell (left image). The predetermined cell is illuminated whilst -1 .2 V vs. Ag/AgCI/3 M NaCI was applied to initiate cleavage of the monolayer directly below the cell (middle image of Figure 16). The electrochemically released predetermined individual cell is recovered through micromanipulation whilst non-illuminated cells remain unaffected (right image of Figure 16).

Example 10: Stability of the ferrocene functionalized surface

By biasing Si at a potential of E > E _FB , the light and dark effect on the electro- catalysis process of the photoanode was studied, and the results presented in Figure 17. The amperometric trace demonstrating the switching on and off of ferrocene-mediated charge transfer from an n-Si electrode to an aqueous solution of Fe(CN) ₆ ^4_ with modulation of the illumination for 200 s each. Under illumination, the photoanodic current is observed as ~ 600.0 μΑ due to the surface tethered ferrocene mediated charge transfer with Fe(CN)6 ^4_ , on the other hand, in the dark, the current is ~ 0.0 μΑ, which indicates the Si is in depletion. Conversely, the photoanodic current is ~ 0.0 μΑ as observed for nonadiyne modified surface, whether in the dark or light. By knowing the surface confined ferrocene from CV and by integrating the amperometric trace, the turn-over for the ferrocene/ferricenium was calculated. The turn-over number for the ferrocene/ferricenium couple is 26.5 s ^_1 . There is a ~ 5 % decreases in current after 6000 s under light.

Example 11 : Mask-free electrochemical polypyrrole writing on n-type Si(100)

Light activated electrochemistry was used to show we could use the strategy for 'writing' features at precise location on the surface of a n-type silicon electrode. This is illustrated schematically in Figure 18. As shown, polymer (surf) can be electrodeposited using monomer via holes-mediated monomer oxidation (reaction 1 ). This was achieved using a 1 ,8-nonadiyne modified n-type silicon electrode (8-12 Ω cm resistivity, -200 μιη thick, with E _FB of -0.34 ± 0.02 V) and electrodepositing polypyrrole from pyrrole monomers. Deposition of polypyrrole by was achieved by poising the silicon electrode at 0.2 V (versus Ag|AgCI) from a solution of pyrrole prepared at the concentration of 5.0 χ 10 ^_1 M in acetonitrile containing 1 .0 * 10 ^_1 M BU ₄ NCIO ₄ under illumination.

An experiment was performed in which an array of polypyrrole was deposited on Si. Each spot of polypyrrole was deposited for 2 s and the Si was biased at 0.24 V vs. Ag|AgCI. The light set up included a supra-band gap illuminator (collimated 642 nm laser) 120. The apparatus used a single lead 125. The laser light was operated at a constant current of 100 mA (- 1 1 mW) and a beam shutter was used to control the illumination of Si precisely. The Si was illuminated from bottom with a distance 500 μηι from each other and FTIR microscopy image was used to characterize the array. See Figure 19.

Extending the capability from dots alone, the next capability developed was to show that images can be drawn on Si by moving the light pointer, illuminating from the backside of the silicon, during the deposition. In the first instance this image was a dot of polypyrrole being deposited for a second followed by a series of lines were drawn on Si where the light travelled at different speed such as 1000, 2000 and 3000 μηπ/ο, respectively. The IR image is shown as a colour gradient at 1 165 cm ^"1 . In the plot of time/s versus current μΑ, the horizontal arrow indicates the "light off" scenario whilst the vertical arrow indicates "light on" scenario. See Figure 20.

After drawing a lines and dot together, a cartoon face was drawn on Si by scanning the light source from bottom. The face is a combination of 2 dots and 2 lines of polypyrrole in which the dot was deposited for 1 s and the line was drawn for 1 s. For lines, the light was travelled for 500 μηι c ^~1 . See Figure 21 .

Example 12: Modulation of apparent formal potential by varying light intensity

We have demonstrated the light switching property of AQ terminated poorly doped, p-type, Si (100) system, and visible light driven AQ redox process has been successfully demonstrated. Figure 22 further shows the light intensity dependence on the cathodic peak for an AQ terminated p-type Si (100) at pH 10.09. The anodic peak potential always stays at around -66 ± 4 mV, while the cathodic peaks as well as apparent formal potential move towards positive potential with the increase of light intensity (see arrow).

Example 13: Light addressable cobalt deposition on AQ terminated silicon surface

Metal deposition on AQ terminated silicon could be realized either via cyclic voltammogram or through chronoamperometry technique. Generally, the diameters of the cobalt dots (Figure 23. a) are much larger than the diameter of the original beam spot (18 μηπ). The approximate diameters under the illumination area for the deposition spots are not equal value, from 79 μηι to 1800 μηπ. The image scanning electron microscope shows the morphology of the cobalt deposited on silicon surface.