A Real-Time Thermal Self-Elimination Method for Static Mode Operated Freestanding Piezoresistive Microcantilever-Based Biosensors.

Here, we provide a method and apparatus for real-time compensation of the thermal effect of single free-standing piezoresistive microcantilever-based biosensors. The sensor chip contained an on-chip fixed piezoresistor that served as a temperature sensor, and a multilayer microcantilever with an embedded piezoresistor served as a biomolecular sensor. This method employed the calibrated relationship between the resistance and the temperature of piezoresistors to eliminate the thermal effect on the sensor, including the temperature coefficient of resistance (TCR) and bimorph effect. From experimental results, the method was verified to reduce the signal of thermal effect from 25.6 µV/°C to 0.3 µV/°C, which was approximately two orders of magnitude less than that before the processing of the thermal elimination method. Furthermore, the proposed approach and system successfully demonstrated its effective real-time thermal self-elimination on biomolecular detection without any thermostat device to control the environmental temperature. This method realizes the miniaturization of an overall measurement system of the sensor, which can be used to develop portable medical devices and microarray analysis platforms.

Direct determination of a small-molecule drug, valproic Acid, by an electrically-detected microcantilever biosensor for personalized diagnostics.

Direct, small-molecule determination of the antiepileptic drug, valproic acid, was investigated by a label-free, nanomechanical biosensor. Valproic acid has long been used as an antiepileptic medication, which is administered through therapeutic drug monitoring and has a narrow therapeutic dosage range of 50-100 µg·mL-1 in blood or serum. Unlike labeled and clinically-used measurement techniques, the label-free, electrical detection microcantilever biosensor can be miniaturized and simplified for use in portable or hand-held point-of-care platforms or personal diagnostic tools. A micromachined microcantilever sensor was packaged into the micro-channel of a fluidic system. The measurement of the antiepileptic drug, valproic acid, in phosphate-buffered saline and serum used a single free-standing, piezoresistive microcantilever biosensor in a thermally-controlled system. The measured surface stresses showed a profile over a concentration range of 50-500 µg·mL-1, which covered the clinically therapeutic range of 50-100 µg·mL-1. The estimated limit of detection (LOD) was calculated to be 45 µg·mL-1, and the binding affinity between the drug and the antibody was measured at around 90 ± 21 µg·mL-1. Lastly, the results of the proposed device showed a similar profile in valproic acid drug detection with those of the clinically-used fluorescence polarization immunoassay.

Improving the binding efficiency of quartz crystal microbalance biosensors by applying the electrothermal effect.

A quartz crystal microbalance (QCM) serving as a biosensor to detect the target biomolecules (analytes) often suffers from the time consuming process, especially in the case of diffusion-limited reaction. In this experimental work, we modify the reaction chamber of a conventional QCM by integrating into the multi-microelectrodes to produce electrothermal vortex flow which can efficiently drive the analytes moving toward the sensor surface, where the analytes were captured by the immobilized ligands. The microelectrodes are placed on the top surface of the chamber opposite to the sensor, which is located on the bottom of the chamber. Besides, the height of reaction chamber is reduced to assure that the suspended analytes in the fluid can be effectively drived to the sensor surface by induced electrothermal vortex flow, and also the sample costs are saved. A series of frequency shift measurements associated with the adding mass due to the specific binding of the analytes in the fluid flow and the immobilized ligands on the QCM sensor surface are performed with or without applying electrothermal effect (ETE). The experimental results show that electrothermal vortex flow does effectively accelerate the specific binding and make the frequency shift measurement more sensible. In addition, the images of the binding surfaces of the sensors with or without applying electrothermal effect are taken through the scanning electron microscopy. By comparing the images, it also clearly indicates that ETE does raise the specific binding of the analytes and ligands and efficiently improves the performance of the QCM sensor.

Detection of the antiepileptic drug phenytoin using a single free-standing piezoresistive microcantilever for therapeutic drug monitoring.

Phenytoin, one of the most widely used antiepileptic drugs, suppresses the abnormal brain activity often seen in seizures. In this study, we report the electrical detection of phenytoin as an antiepileptic medication with a narrow therapeutic dosage range to which therapeutic drug monitoring (TDM) is applied. The measurement technique used an electrical detection of a piezoresistive microcantilever biosensor. This label-free, electrically measured microcantilever can be miniaturized in order to be portable for point-of-care, personal diagnosis or for personalized therapeutic drug monitoring. The miniaturized piezoresistive microcantilever was fabricated by micro-electro-mechanical system processes, and was integrated into a microfluidic channel with a system for label-free detection. The microcantilever biosensor was approved for the detection of phenytoin in solutions of deionized water and 100% fetal bovine serum. A linear profile in a drug-concentration range of 10-80 µg/mL was detected, with the signal resolution being about 0.005 Ω. The concentration sensitivity was 2.94×10(-6) (µg/mL)(-1). The binding affinity (KD) was calculated to be 58 µg/mL. The results of the present piezoresistive microcantilever biosensors showed a solid correlation of phenytoin drug detection with that in the clinically used fluorescence polarization immunoassay (FPIA).

Electrical detection of C-reactive protein using a single free-standing, thermally controlled piezoresistive microcantilever for highly reproducible and accurate measurements.

This study demonstrates a novel method for electrical detection of C-reactive protein (CRP) as a means of identifying an infection in the body, or as a cardiovascular disease risk assay. The method uses a single free-standing, thermally controlled piezoresistive microcantilever biosensor. In a commonly used sensing arrangement of conventional dual cantilevers in the Wheatstone bridge circuit, reference and gold-coated sensing cantilevers that inherently have heterogeneous surface materials and different multilayer structures may yield independent responses to the liquid environmental changes of chemical substances, flow field and temperature, leading to unwanted signal disturbance for biosensing targets. In this study, the single free-standing microcantilever for biosensing applications is employed to resolve the dual-beam problem of individual responses in chemical solutions and, in a thermally controlled system, to maintain its sensor performance due to the sensitive temperature effect. With this type of single temperature-controlled microcantilever sensor, the electrical detection of various CRP concentrations from 1 µg/mL to 200 µg/mL was performed, which covers the clinically relevant range. Induced surface stresses were measured at between 0.25 N/m and 3.4 N/m with high reproducibility. Moreover, the binding affinity (KD) of CRP and anti-CRP interaction was found to be 18.83 ± 2.99 µg/mL, which agreed with results in previous reported studies. This biosensing technique thus proves valuable in detecting inflammation, and in cardiovascular disease risk assays.

Binding kinetics of grouper nervous necrosis viruses with functionalized antimicrobial peptides by nanomechanical detection.

We report the binding kinetics of fish-infected grouper nervous necrosis viruses (NNV) and selected antimicrobial peptides (AMPs) by nanomechanical detection. AMPs, the vital member in an innate immunity, are promising candidates in the fight against pathogens due to their broad range of antimicrobial activity and low toxicity. Grouper NNV primarily cause mass mortality of many marine cultured fish species, and two selected AMPs in this study were found to inhibit viruses by agglutinating its virions to form aggregates. The binding activity of NNVs with functionalized AMPs onto a sensing microcantilever yielded induced surface stresses, indicating high binding strength of molecular interaction. The binding affinity and kinetic rate constants of molecular recognition events calculated for NNV-AMP(TH1-5) compared to NNV-AMP(cSALF) were found to be 2.1-fold and 4.43-fold, respectively, indicating TH1-5 effectively bind with NNV more than cSALF. Moreover, a microscopic X-ray photoelectron spectroscopy technique was employed for further validation of pre- and post-NNV binding onto peptides-functionalized sensing surface. An increase in the spectrum and intensity of the P 2p and N 1s elements for the post-NNV binding was clearly shown to ensure the existence of phosphate groups and nitrogen-containing ring structures of specific NNV-TH1-5 interaction. Therefore, the microcantilever biosensing technique provides a potential and useful screening of AMPs for affinity to NNVs.

Laser ablation of silicon using a Bessel-like beam generated by a subwavelength annular aperture structure.

Using a femtosecond laser incident to an oxide-metal-oxide film engraved with a subwavelength annular aperture (SAA) structure, we generated a Bessel-like beam to ablate silicon. Experimental results show that the silicon can be ablated with a 0.05 J/cm(2) input ablation threshold at 120 fs pulse duration. We obtained a surface hole possessing a diameter less than 1 µm. Optical performance, including depth-of-focus and focal spot of the SAA structure, were simulated using finite-different time-domain calculations. We found that a far-field laser beam propagating through a SAA structure possesses a submicrometer focal spot and high focus intensity. Our method can be easily adopted for surface machining in microfabrication applications.

Cell docking, movement and cell-cell interactions of heterogeneous cell suspensions in a cell manipulation microdevice.

This study demonstrates a novel cell manipulation microdevice for cell docking, culturing, cell-cell contact and interaction by microfluidic manipulation of heterogeneous cell suspensions. Heterogeneous cell suspensions include disparate blood cells of natural killer cells and leukemia cancer cells for immune cell transplantation therapy. However, NK cell alloreactivity from different healthy donors present various recovery response levels. Little is still known about the interactions and cytotoxicity effects between donor NK cells and recipient cancer cells. The cell-based micro device first showed the capability of cell docking, movement, contact and cell-cell interaction with respect to cell cytotoxicity of NK cells against cancer cells. With various flow tests for live cell loading, flow rates of 10 µL/h were chosen for injection in the central and side flows such that both types of suspension cells could be gently docked at the gap structure in a reaction zone. The trapping number of particles and cells was linearly proportional to the gap length. Finally, the cytotoxicity of around 40% was found to be similar in the case of dilute cells and a large cell population. As a result, the cell manipulation microdevice has been validated for live suspensions of natural killer and cancer cells, and exhibited the capability to measure the cytotoxicity of dilute cell suspensions.

A wireless bio-MEMS sensor for C-reactive protein detection based on nanomechanics.

A quick wireless label-free detection of disease-related C-reactive proteins (CRPs) using a 200-microm-long microelectromechanical systems (MEMS) microcantilever housed in a 7 x 7 mm(2) reaction chamber with a safe reusable feature is reported. The assay time ranges from about 30 min to 3 h, depending on accuracy. The deflection of the microcantilever due to specific CRP-antiCRP binding is detected using a position-sensitive detector. The converted bio-signal is transmitted by a custom designed wireless amplitude-shift-keying (ASK) transceiver IC fabricated in a 0.18 microm CMOS process. CRP concentrations from 1 microg/mL to 500 microg/mL can be detected. A 0.2-Hz 1-V ac signal instead of traditional bases/acids is applied to the bio-MEMS sensor to unbind the CRP from the microcantilever for reusability.

Effect of subwavelength annular aperture diameter on the nondiffracting region of generated Bessel beams.

A subwavelength annular aperture (SAA) made on metallic film and deposited on a glass substrate was fabricated by electron-beam lithography (EBL) and which was followed by a metal lift-off process to generate a long propagation range Bessel beam. We propose tuning the focal length and depth of focus (DOF) by changing the diameter of the SAA. We used finite-difference time domain (FDTD) simulations to verify our experimental data. We found that the position of the Bessel Beam focus spot (i.e. focal length) will be farther away from the SAA plane as the diameter of the SAA increases. In addition, the depth of focus (DOF) which is the length of the Bessel beam non-diffracting area, also increases as the diameter of the SAA expands.

Dynamic response of glucagon/anti-glucagon pairs to pulling velocity and pH studied by atomic force microscopy.

We used atomic force microscopy (AFM) to measure the unbinding force between antigen coupled to an AFM tip and antibody coated on the substrate surface. Dynamic responses of glucagon/anti-glucagon pairs with multiple pull-off steps to pH and pulling velocity were studied by AFM. Force-distance curves of a specific glucagon-anti-glucagon interaction system with mono-, di-, and multi-unbinding events were recorded, which may be attributed to a single, sequential or multiple breaking of interacting bond(s) between glucagon and anti-glucagon. We studied the dynamic response of glucagon-anti-glucagon pairs to various pulling velocities (16.7-166.7 nm/s). It was found that the mean value of the unbinding force was shifted toward higher values with increasing pulling velocity at each pH. This indicates that the friction force between glucagon and anti-glucagon may contribute to the unbinding force. Moreover, the dynamic response of glucagon-anti-glucagon pairs to pH (4-10) with different pulling velocities was studied. Within the acid range, the bond strength between the glucagon/anti-glucagon complex showed a rapid increase from pH 4 to 7 and reached a maximum (256.4+/-48.9 pN at 166.7 nm/s) at neutrality, followed by a sharp decrease with increasing pH (pH 7-10). This could be attributed to the conformational change that occurred in glucagon when the pH value in solution was varied from the reference level at neutrality. This study demonstrated that the pH dependence of multiple antigen-antibody bond-rupture forces could be measured by a force-based AFM biosensor. Unraveling the relationship between inter-molecular force and intra-molecular conformational change in acid, neutral, and alkaline environments may provide new directions for future application of force measurements by AFM in proteomics or in the development of a clinical cantilever-based mechanical biosensor.

Atomic force microscopy: determination of unbinding force, off rate and energy barrier for protein-ligand interaction.

Recently, atomic force microscopy (AFM) based force measurements have been applied biophysically and clinically to the field of molecular recognition as well as to the evaluation of dynamic parameters for various interactions between proteins and ligands in their native environment. The aim of this review is to describe the use of the AFM to measure the forces that control biological interaction, focusing especially on protein-ligand and protein-protein interaction modes. We first considered the measurements of specific and non-specific unbinding forces which together control protein-ligand interactions. As such, we will look at the theoretical background of AFM force measurement curves for evaluating the unbinding forces of protein-ligand complexes. Three AFM model dynamic parameters developed recently for use in protein-ligand interactions are reviewed: (i) unbinding forces, (ii) off rates, and (iii) binding energies. By reviewing the several techniques developed for measuring forces between biological structures and intermolecular forces in the literature, we show that use of an AFM for these applications provides an excellent tool in terms of spatial resolution and lateral resolution, especially for protein-protein and protein-ligand interactions.

Measurement of dimensions of pentagonal doughnut-shaped C-reactive protein using an atomic force microscope and a dual polarisation interferometric biosensor.

In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. A single pentagonal structure was directly visualized by AFM, and quantitative measurements of the dimensions of the protein were provided. The average height calculated for each pentagonal CRP particle was approximately 3.03+/-0.37 nm, which basically corresponds to that (36 A in protomer diameter) previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, a experiment using dual polarization interferometric (DPI) as a biosensor was then performed, and the average monolayer thickness value (3.18+/-0.43 nm) that was calculated basically corresponds to that obtained from the experimental value (3.03+/-0.37 nm) of the height measured by an AFM method for CRP. Further investigations will be performed to study the surface ultrastructure of a single pentagonal CRP molecule, and for this purpose a CRP sample (at low concentration) was scanned in vacuum by AFM. The higher-resolution images clearly revealed the presence of doughnut-shaped CRP molecules. In addition, phase images of CRP molecules were captured simultaneously with their height images, and the lateral dimensions of the doughnut-shaped CRP molecules were then measured. It was found that the average values calculated for the outer diameter (11.13+/-1.47 nm) and pore diameter (3.52+/-0.42 nm) are respectively close to those (102 A in outer diameter and 30 A in pore diameter) previously obtained from the structure of CRP determined by X-ray crystallography. This study represents the first direct characterization of the surface ultrastructure and dimensional measurement of the CRP molecule on the sensor chip.

Fabrication of polymer microlens arrays using capillary forming with a soft mold of micro-holes array and UV-curable polymer.

This paper reports a simple and effective method to fabricate microlens arrays with the ultraviolet-curable resins, and a soft mold of micro-holes array. During capillary forming operation, the surface of the soft mold of micro-holes array is being pressed against the ultraviolet-curable resin layer coated on the plastic substrate. An array of convex lense can be formed in the circular holes of the soft mold due to the capillary filling and surface tension. The microlens arrays have smooth surface and uniform focusing function. The shape and height of micolens can be controlled with a proper combination of pressing pressure, pressing duration and UV curing dose. This technique shows great potential for fabricating polymer microlens arrays with high productivity and low cost.

Surface ultrastructure of SARS coronavirus revealed by atomic force microscopy.

Atomic force microscopy has been used to probe the surface nanostructures of severe acute respiratory syndrome coronavirus (SARS-CoV). Single crown-like virion was directly visualized and quantitative measurements of the dimensions for the structural proteins were provided. A corona of large, distinctive spikes in the envelope was measured after treatment with hydroxyoctanoic acid. High-resolution images revealed that the surface of each single SARS-CoV was surrounded with at least 15 spherical spikes having a diameter of 7.29 +/- 0.73 nm, which is in close agreement with that of S glycoproteins earlier predicted through the genomes of SARS-CoV. This study represents the first direct characterization of the surface ultrastructures of SARS-CoV particles at the nanometre scale and offers new prospects for mapping viral surface properties.

Determination of binding constant of DNA-binding drug to target DNA by surface plasmon resonance biosensor technology.

The experimental determination of the binding constant of a drug for its target molecule is of considerable importance. It is a basic experimental parameter in a variety of studies, such as the prediction of drug efficiency, or in the pharmacokinetic drug interaction. DNA-binding drugs have been reported to be able to interfere in a sequence dependent manner with biological functions such as topoisomerase activity, restriction of enzyme cleavage of DNA, protein-DNA interactions and the activity of transcription factors, leading to alteration of gene expression. This effect could have important practical application in the experimental therapy of human pathologies, including neoplastic diseases and viral, or microbial infections. The assessment of the biological activity of DNA-binding drugs by polymerase chain reaction, footprinting, gel retardation and in vitro transcription studies was recently reported. However, most of these techniques are steady-state methodologies and therefore are not suitable for an easy determination of the binding activity of DNA-binding drugs to target DNA and the stability of drugs-DNA complexes. Direct real-time observation and measurement of the interaction between DNA-binding drug and target DNA sequence is a subject of interest for drug discovery and development. The recent development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions of DNA-binding drugs with target DNA elements in real-time. The present review is designed to indicate the theoretical background of SPR-based biosensor technology as well as to present the great variety of measurements and modes of interaction kinetics that can be performed with these techniques. In addition, some of the most recent studies in determining the binding constant and stoichiometry of DNA-binding drugs to target DNA with SPR technology are reviewed and the available theoretical aspects necessary for the comprehension of the experiments are provided.