Scanning Photo-induced Impedance Microscopy Research Articles

Light-Addressable Electrochemical Sensors toward Spatially Resolved Biosensing and Imaging Applications

The light-addressable electrochemical sensor (LAES) is a recently emerged bioanalysis technique combining electrochemistry with the photoelectric effect in a semiconductor. In an LAES, a semiconductor substrate is illuminated locally to generate charge carriers in a well-defined area, thereby confining the electrochemical process to a target site. Benefiting from the unique light addressability, an LAES can not only detect multiple analytes in parallel within a single sensor plate but also act as a bio(chemical) imaging sensor to visualize the two-dimensional distribution of specific analytes. An LAES usually has three working modes: a potentiometric mode using light-addressable potentiometric sensors (LAPS) and an impedance mode using scanning photoinduced impedance microscopy (SPIM), while an amperometric mode refers to light-addressable electrochemistry (LAE) and photoelectrochemical (PEC) sensing. In this review, we describe the detection principles of each mode of LAESs and the concept of light addressability. In addition, we highlight the recent progress and advance of LAESs in spatial resolution, sensor system design, multiplexed detection, and bio(chemical) imaging applications. An outlook on current research challenges and future prospects is also presented.

Scanning Electrochemical Photometric Sensors for Label-Free Single-Cell Imaging and Quantitative Absorption Analysis.

A new photoelectrochemical imaging method termed scanning electrochemical photometric sensor (SEPS) is proposed in this work. It was derived from light-addressable potentiometric sensor (LAPS) and scanning photoinduced impedance microscopy (SPIM) using a front-side laser illumination at a field-effect structure. When the laser beam scans across the sensor substrate, local photocurrent changes at inversion due to the light absorption of analytes can be recorded. It will be shown that SEPS could be used for label-free living cell imaging with micro-resolution as well as real-time quantitative absorption analysis, which would broaden the applications of traditional LAPS/SPIM from potentiometric/impedance measurements to local optical analysis.

A Gas‐Sensitive SPIM Sensor for Detection of Ethanol Using SnO2 as Sensing Element

In this study, a gas‐sensitive scanning photo‐induced impedance microscopy (SPIM) sensor using tin dioxide (SnO2) as sensing element is proposed, which detects the local impedance change on the sensor surface in response to ethanol vapor. The impedance change of the SnO2 film is detected by monitoring the AC photocurrent signal induced by a laser beam. This measurement can be done in a spatially resolved manner by using a scanning laser beam and two‐dimensional maps are obtained thanks to the light‐addressability of SPIM. This technique is expected to be applied to the visualization of gas distributions on the sensing surface.

LAPS and SPIM Imaging Using ITO-Coated Glass as the Substrate Material.

Light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) use photocurrent measurements for spatiotemporal imaging of ion concentrations, electrical potentials, and impedance. In this work, ITO-coated glass was confirmed to produce photocurrents at anodic potentials with 405 nm diode laser illumination. Therefore, it was developed as a low cost and robust substrate material for LAPS and SPIM imaging compared to traditional expensive ultrathin Si substrates. ITO showed good ac photocurrent and pH response without surface modification and insulator. Local photocurrents were produced by scanning a focused laser beam across the sample, which proved the light addressability of ITO-coated glass. With a high-impedance PMMA dot deposited onto the ITO as a model system, a lateral resolution of about 2.3 μm was achieved.

Biological imaging using light-addressable potentiometric sensors and scanning photo-induced impedance microscopy.

Light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) use photocurrent measurements at electrolyte-insulator-semiconductor substrates for spatio-temporal imaging of electrical potentials and impedance. The techniques have been used for the interrogation of sensor arrays and the imaging of biological systems. Sensor applications range from the detection of different types of ions and the label-free detection of charged molecules such as DNA and proteins to enzyme-based biosensors. Imaging applications include the temporal imaging of extracellular potentials and dynamic concentration changes in microfluidic channels and the lateral imaging of cell surface charges and cell metabolism. This paper will investigate the current state of the art of the measurement technology with a focus on spatial and temporal resolution and review the biological applications, these techniques have been used for. An outlook on future developments in the field will be given.

Electrochemical Imaging of Copper Contamination on Self-Assembled Organic Monolayer Modified Silicon Surfaces Following a ‘Click’ Reaction with Light-Addressable Potentiometric Sensors and Scanning Photo-Induced Impedance Microscopy

Light-addressable potentiometric sensors (LAPS)1 and scanning photo-induced impedance microscopy (SPIM)2, both of which are based on photocurrent measurements at electrolyte /insulator /semiconductor (EIS) field-effect structures can be used to detect local surface electrical potentials and impedance, respectively. While LAPS measures local potential changes by recording the shift of the photocurrent-voltage (I-V) curve along the voltage axis, SPIM records changes in the maximum photocurrent while the EIS structure is biased towards inversion. Both techniques have been extensively applied for chemical ion sensing, biosensing, and the investigation of cell metabolism, extracellular potentials, cell surface charge and impedance. Importantly, they have been reported to be highly sensitive to the surface contamination on self-assembled organic monolayers3. In this work, a copper (I) catalyzed azide alkyne cycloaddition (CuAAC) reaction, combined with microcontact printing was employed successfully to pattern alkyne-terminated self-assembled organic monolayer modified silicon surfaces (Figure 1a). Despite the absence of a copper peak in XPS spectra, copper contamination was found and visualized using LAPS and SPIM, after the ‘click’ modified silicon surfaces were rinsed with hydrochloric acid solution (Figure 1b), which has been frequently used to remove copper residues in the past. Different strategies for avoiding copper contamination, including the use of bulky chelators for the copper (I) catalyst and rinsing with different reagents, were tested. Only cleaning of the silicon surfaces with an ethylenediaminetetraacetic acid (EDTA) solution after the ‘click’ modification proved to be an effective method as confirmed by LAPS and SPIM results, which showed the desirable electrical properties (surface charge and impedance) of an organic monolayer (Figure 1c). 1. Hafeman, D. G.; Parce, J. W.; McConnell, H. M., LIGHT-ADDRESSABLE POTENTIOMETRIC SENSOR FOR BIOCHEMICAL SYSTEMS. Science 1988,240, (4856), 1182-1185. 2. Krause, S.; Talabani, H.; Xu, M.; Moritz, W.; Griffiths, J., Scanning photo-induced impedance microscopy - an impedance based imaging technique. Electrochim. Acta 2002,47, (13-14), 2143-2148. 3. Wang, J.; Wu, F.; Watkinson, M.; Zhu, J. Y.; Krause, S., "Click" Patterning of Self-Assembled Monolayers on Hydrogen-Terminated Silicon Surfaces and Their Characterization Using Light-Addressable Potentiometric Sensors. Langmuir 2015, 31, (35), 9646-9654. Figure 1

Image Detection of Yeast Saccharomyces Cerevisiae By Light-Addressable Potentiometric Sensors (LAPS)

: The Light-Addressable Potentiometric Sensors (LAPS) and Scanning Photo-induced Impedance Microscopy (SPIM) was developed for the image detection of multilayer yeast Saccharomyces cerevisiae. Low gelling temperature agarose gel was shown to be an effective immobilization reagent for yeast cells in LAPS measurements without background signal. The images were produced by the photocurrent differences between the area where yeast cells were attached and the 1, 8-nonadiyne modified silicon on sapphire (SOS) surface, which were induced by the surface negative charge and local impedance of yeast cells. Keywords: Light-Addressable Potentiometric Sensors, cell image, Saccharomyces cerevisiae Introduction LAPS has been used successfully for the chemical imaging of specific ions, DNA detection, monitoring of enzymatic reactions, microorganisms, activities and impedance of cells.[1] The resolution was about 10-100 µm generally using LED arrays as the light source. We optimized the setup by scanning a focused laser beam across the sample thereby exciting local photocurrents and achieved submicrometer resolution[2], which we are now proposing to use for imaging living cells with high resolution. Here we report the LAPS image detection of multilayer yeast Saccharomyces cerevisiae. Results and Discussion Multiple yeast cells (4 × 107cells/mL, 0.2 µL) were immobilized onto the 1, 8-nonadiyne modified SOS surface by low melting agarose gel. Pure agarose gel was also dropped onto the same sample surface as the control experiment (Figure 1(A)). Figure 1 (B) showed the LAPS image of yeast-agarose gel on a 1, 8-nonadiyne modified SOS surface at 0.7 V using a 405 nm laser. As control experiment, the pure agarose gel did not show a difference in the LAPS signal with the 1, 8-nonadiyne modified SOS. The maximum photocurrents on the yeast attached area were lower than that on the blank monolayer surface and the agarose gel attached area, indicating an increase of the local impedance. After normalizing, the I-V curves on the yeast attached area shifted by +90 mV compared to the blank monolayer surface and the agarose gel attached area, corresponding to the negative charge of the yeast cells in pH 7.4 PBS solution. Figure captions Figure 1. Yeast-agarose gel on a 1,8-nonadiyne modified SOS surface; (A) Optical microscope image of (a) pure agarose gel and (b) yeast-agarose gel; (B) LAPS photocurrent image measured at 0.7 V; (C) I-V curves on yeast-agarose gel, blank surface, and agarose gel; (D) normalised I-V curves and potential shift . Conclusions We report the LAPS image of multilayers yeast cells. The signals of yeast cells came from the negative surface charge and the local impedance. In the future we hope to achieve single cell LAPS images.

Copper Contamination of Self-Assembled Organic Monolayer Modified Silicon Surfaces Following a “Click” Reaction Characterized with LAPS and SPIM

A copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction combined with microcontact printing was used successfully to pattern alkyne-terminated self-assembled organic monolayer-modified silicon surfaces. Despite the absence of a copper peak in X-ray photoelectron spectra, copper contamination was found and visualized using light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) after the "click"-modified silicon surfaces were rinsed with hydrochloric acid (HCl) solution, which was frequently used to remove copper residues in the past. Even cleaning with an ethylenediaminetetraacetic acid (EDTA) solution did not remove the copper residue completely. Different strategies for avoiding copper contamination, including the use of bulky chelators for the copper(I) catalyst and rinsing with different reagents, were tested. Only cleaning of the silicon surfaces with an EDTA solution containing trifluoroacetic acid (TFA) after the click modification proved to be an effective method as confirmed by LAPS and SPIM results, which showed the expected potential shift due to the surface charge introduced by functional groups in the monolayer and allowed, for the first time, imaging the impedance of an organic monolayer.

The effect of gold nanoparticles on the impedance of microcapsules visualized by scanning photo-induced impedance microscopy

Polyelectrolyte microcapsules have attracted great interest in drug delivery applications, and microcapsules modified with gold nanoparticles have been used in this way with triggered release when a laser can be used to remotely open shells through light-induced local heating. The electrical impedance of unmodified microcapsules has been studied due to its implications for their permeability, however, the impedance of functionalised microcapsules has not yet been investigated. Herein, the impedance of microcapsules modified with gold nanoparticles was studied for the first time. It was shown that the modification of microcapsules with gold nanoparticles leads to a much greater impedance than would be expected from the increase in the thickness caused by the presence of a layer of gold nanoparticles alone. The impedance of gold nanoparticle modified capsules was measured using scanning photo-induced impedance microscopy (SPIM), which is based on photocurrent measurements at an electrolyte-insulator-semiconductor (EIS) field-effect structure. High resolution and good sensitivity were achieved using a two-photon effect for charge carrier excitation and organic monolayer modified silicon-on-sapphire (SOS) as the SPIM substrate. SPIM allowed impedance imaging of collapsed microcapsules with unprecedented detail. SPIM images of capsules labelled with gold nanoparticles (AuNPs) showed a good agreement with the corresponding optical images, including the creases resulting from the collapse of the hollow shells. The significant increase in impedance caused by the impregnation with AuNPs was also verified by conductive Atomic Force Microscopy (C-AFM) measurements in the dry state.

High resolution LAPS and SPIM

The resolution of photocurrent measurements at field-effect capacitors as used in light-addressable potentiometric sensors (LAPS) and scanning photo-induced impedance microscopy (SPIM) has been investigated using silicon on sapphire (SOS) substrates illuminated at different wavelengths. Using a two-photon effect in silicon ( λ = 1250 nm) to generate the photocurrent, genuine submicrometer resolution has been demonstrated for LAPS and SPIM. Improved sensitivity for both LAPS and SPIM was obtained using a 6.7 nm thick gate oxide on SOS anodically grown in 0.1 M HCl.

Repair of thin thermally grown silicon dioxide by anodic oxidation

Anodic oxidation in 0.1 M HCl followed by a post-rapid thermal annealing process has been used to repair defects existing in thin thermally grown oxide layers (3 and 6 nm) on a p-type silicon substrate. The improved quality of the insulator layer is particularly useful for applications that require large gate areas in Metal–Insulator–Semiconductor (MIS) or Electrolyte–Insulator–Semiconductor (EIS) devices such as Light-Addressable Potentiometric Sensors (LAPS) and Scanning Photo-induced Impedance Microscopy (SPIM). Different methods have been used to characterize the oxide. High-frequency capacitance–voltage curves and ac impedance spectra showed that there was no significant change of the oxide thickness after repair, and the number of the interface states of the oxide was increased for both types of samples. Ramped voltage stress (RVS) measurements of Metal–Oxide–Semiconductor (MOS) structures with gate electrodes 2 mm in diameter showed leakage currents of 0.75 nA cm −2 for the repaired and annealed 3 nm thick oxide and 1.31 nA cm −2 for the repaired and annealed 6 nm thick oxide at accumulation voltage. XPS measurements confirmed that there was no change of the oxide thickness and no contamination with other ionic species after repair. AFM results showed a good agreement with the other characterization methods.

Biosensor Arrays Based on the Degradation of Thin Polymer Films Interrogated by Scanning Photoinduced Impedance Microscopy

Disposable sensors based on the degradation of thin films as a result of an enzymatic reaction have been developed into efficient enzyme detectors. Film degradation has traditionally been monitored using surface plasmon resonance (SPR), quartz crystal microbalance (QCM), or classical ac impedance measurements. The enzyme detection principle has now been integrated with an array technology derived from a recently developed impedance imaging technique, scanning photoinduced impedance microscopy (SPIM). SPIM is based on photocurrent measurements at field-effect structures. The material under investigation is commonly deposited onto a semiconductor-insulator substrate. In this work, field-effect capacitors were replaced by hydrogenated amorphous silicon (a-Si:H) n-i-p photodiode structures, which have recently been shown to be suitable for SPIM measurements with good lateral resolution. To demonstrate the feasibility of SPIM for the characterization of biosensor arrays, polymer dots of the inert polymer cellulose acetate and an alpha-chymotrypsin-sensitive poly(ester amide) were deposited onto a-Si:H n-i-p/SiO2 structures and their enzymatic degradation was monitored using a laser scanning setup.

Scanning Photoinduced Impedance Microscopy Using Amorphous Silicon Photodiode Structures

Scanning photoinduced impedance microscopy (SPIM) is an impedance imaging technique, which is based on photocurrent measurements at electrolyte-insulator-semiconductor (EIS) and metal-insulator-semiconductor (MIS) field-effect structures. The material to be investigated has to be deposited on top of the insulator (E/I or M/I interface). The lateral resolution of SPIM is limited by the lateral diffusion of minority charge carriers. Therefore, it would be advantageous if semiconductors with a short diffusion length of charge carriers such as amorphous silicon could be employed. However, field-effect capacitors fabricated using amorphous silicon suffered from a large number of interface states, high leakage currents through the insulator, and small photocurrents. In this work, field-effect capacitors were replaced by amorphous hydrogenated silicon photodiode structures (a-Si:H p-i-n/SiO2 or n-i-p/SiO2) as this was expected to result in higher photocurrents and eliminate the necessity of a high-quality insulator. The photodiode structures were shown to be suitable for SPIM measurements. The resolution of photocurrent measurements was found to depend strongly on the frequency of the modulated light and the doping concentration of the amorphous silicon layer closest to the insulator. An equivalent circuit model was developed to simulate this behavior.

Scanning Photo-Induced Impedance Microscopy—Resolution studies and polymer characterization

Scanning Photo-Induced Impedance Microscopy (SPIM) is an impedance imaging technique that is based on photocurrent measurements at field-effect structures. The material under investigation is deposited onto a semiconductor–insulator substrate. A thin metal film or an electrolyte solution with an immersed electrode serves as the gate contact. A modulated light beam focused into the space charge region of the semiconductor produces a photocurrent, which is directly related to the local impedance of the material. The absolute impedance of a polymer film can be measured by calibrating photocurrents using a known impedance in series with the sample. Depending on the wavelength of light used, charge carriers are not only generated in the focus but also throughout the bulk of the semiconductor. This can have adverse effects on the lateral resolution. Two-photon experiments were carried out to confine charge carrier generation to the space charge layer. The lateral resolution of SPIM is also limited by the lateral diffusion of charge carriers in the semiconductor. This problem can be solved by using thin silicon layers as semiconductor substrates. A resolution of better than 1 μm was achieved using silicon on sapphire (SOS) substrates with a 1 μm thick silicon layer.

High resolution LAPS using amorphous silicon as the semiconductor material

The lateral resolution of photocurrent techniques such as light-addressable potentiometric sensors (LAPS) or scanning photo-induced impedance microscopy (SPIM) is limited by the properties of the semiconductor material used. We investigated metal-insulator-semiconductor (MIS) structures based on amorphous silicon (a-Si) prepared as a thin layer on transparent glass substrates. It was shown that a sub-micrometer resolution can be achieved for this material, which is much better than the results for single crystalline Si. Some limitations caused by light scattering in the structure were observed.

Scanning photo-induced impedance microscopy—an impedance based imaging technique

A.C. impedance spectroscopy is a valuable tool in the characterisation of electrochemical systems and new materials. However, the results obtained using this method are always surface averaged. A new technique, which is suitable for imaging the complex impedance of electrochemical and solid-state systems with good spatial resolution, has been developed. It is based on photocurrent measurements at field effect structures. A semiconductor–insulator structure serves as the substrate for the film under investigation. A pulsed and focused light beam scanned across the sample surface results in photocurrents, which provide information about the local distribution of the dielectric properties of the film. Thin films of poly methyl methacrylate were investigated as a model system. Absolute values of the impedance could be calculated from the photocurrent measurements using a simple calibration procedure. Good spatial resolution was achieved by using a thin epitaxial film of silicon as the semiconductor substrate.

Photocurrent measurements for laterally resolved interface characterization.

A miniaturized optical set-up based on a CD-ROM player optic was developed for LAPS (light addressable potentiometric sensors). A focus of 2.6 microm was achieved using this easy to handle device. The lateral resolution of LAPS measurements can be improved by using GaAs as the semiconductor material instead of Si. The diffusion length of the minority charge carriers was determined to be smaller than 3.1 microm. A new method called SPIM (scanning photo-induced impedance microscopy) is described. Using this technique, the impedance of thin films can be measured with lateral resolution.