Raj Mutharasan

Biosensing using dynamic-mode cantilever sensors: A review

Current progress on the use of dynamic-mode cantilever sensors for biosensing applications is critically reviewed. We summarize their use in biosensing applications to date with focus given to: cantilever size (milli-, micro-, and nano-cantilevers), their geometry, and material used in fabrication. The review also addresses techniques investigated for both exciting and measuring cantilever resonance in various environments (vacuum, air, and liquid). Biological targets that have been detected to date are summarized with attention to bio-recognition chemistry, surface functionalization method, limit of detection, resonant frequency mode type, and resonant frequency measurement scheme. Applications published to date are summarized in a comprehensive table with description of the aforementioned details including comparison of sensitivities. Further, the general theory of cantilever resonance is discussed including fluid-structure interaction and its dependence on the Reynolds number for Newtonian fluids. The review covers designs with frequencies ranging from ∼1 kHz to 10 MHz and cantilever size ranging from millimeters to nanometers. We conclude by identifying areas that require further investigation.

In situ cell detection using piezoelectric lead zirconate titanate-stainless steel cantilevers

We have investigated piezoelectric lead zirconate titanate (PZT)-stainless steel cantilevers as real-time in-water cell detectors using yeast cells as a model system. Earlier studies have shown that mass changes of a cantilever can be detected by monitoring the resonance frequency shift. In this study, two PZT-stainless steel cantilevers with different sensitivities were used to detect the presence of yeast cells in a suspension. The stainless steel cantilever tip was coated with poly-L-lysine that attracted yeast cells from the suspension, and immobilized them on the cantilever surface. After immersing the poly-L-lysine coated tip in a yeast suspension, the flexural resonance frequency of the cantilever was monitored with time. The flexural resonance frequency decreased with time in agreement with the optical micrographs that showed increasing amount of adsorbed yeast cells with time. The resonance frequency shifts are further shown to be consistent with both the mass of immobilized cells on the poly-L-l...

Feasibility of aluminium nitride formation in aluminum alloys

Abstract The feasibility of forming aluminum nitride by in situ reactive nitrogen gas injection into molten aluminum alloys has been evaluated both analytically and experimentally over the temperature range from 700 to 1500°C. It is shown that aluminum nitride can be melt formed in the presence of Mg and Si, with nitrogen and/or ammonia as the reactive gases at temperature above 1100°C. In this role, magnesium serves as a catalyst. Magnesium niride is first formed in the vapor phase by the reaction of vaporized magnesium and nitrogen gas, followed by incorporation of magnesium nitride particles into the molten aluminum. Via an in situ substitution reaction, aluminum nitride forms between magnesium nitride and aluminum. Up to 17 wt.% aluminum nitride in an aluminum alloy has been formed with an average reinforcement size of 3 μm. The potential for this process permits economical liquid phase processing of aluminum nitride-aluminum metal matrix composite with nitrogen gas injection for structural, thermal and wear applications.

Sample Preparation-Free, Real-Time Detection of microRNA in Human Serum Using Piezoelectric Cantilever Biosensors at Attomole Level

A sensitive, selective, sample preparation-free method for near real-time detection of microRNA in buffer and human serum is given using gold (Au)-coated dynamic piezoelectric cantilever sensors. Sensor response to thiolated DNA probe chemisorption, hsa-let-7a hybridization, labeled-DNA hybridization, and Au nanoparticle-functionalized DNA hybridization was monitored continuously in flowing liquid samples using custom flow-cells. The assay showed successful detection of target let-7a with a dynamic range spanning 6 orders of magnitude (10 fM-1 nM) with a limit of detection of less than 10 attomoles (∼4 fM). The serum background had negligible effect on sensitivity relative to the results obtained in the buffer due to reduction in nonspecific binding caused by continuous sensor vibration. Both hybridization and nonspecific binding reduction were confirmed using fluorescence-based assays to support sensor-based results. The sensor-based method demonstrated excellent selectivity for the microRNA target in comparison with similar microRNA differing by only a single nucleotide (hsa-let-7c) and random microRNA sequences. Au nanoparticle-based amplification of sensor response was investigated and led to an order of magnitude improvement in the detection limit and a 128% amplification of sensor response over the entire dynamic range. Au nanoparticle amplification was verified by scanning electron microscopy. The cantilever sensor-based microRNA assay provides competitive sensitivity with current microRNA detection methods and has the advantage of requiring no sample preparation, even when working with biological samples that contain a complex background.

Fabrication of biconical tapered optical fibers using hydrofluoric acid

Abstract An easy to implement procedure for etching silica fibers in biconical form useful in sensing applications is described. A simple etching reactor was developed to obtain reproducible tapers of desired diameter and length. An approach for on-line monitoring of etching using a commonly used fluorometer is demonstrated. A mathematical model describing the light power transmission is proposed, and is validated using experimental data. The data and the model indicate that the diameter of the silica fiber decreases linearly with time with hydrofluoric acid (HF, 49.5% w/w) used as etchant at room temperature. The observed etching rate was 0.0023±0.00019 s −1 , which was repeatable using the procedure developed in this study. Method to arrest etching and subsequent preservation of the small diameter taper in mildly alkaline solution was found to be successful.

Rapid and sensitive immunodetection of Listeria monocytogenes in milk using a novel piezoelectric cantilever sensor

Listeria monocytogenes (LM), an important food-borne pathogen that has a high mortality rate (≈ 30%), was successfully detected within an hour at the infection dose limit of 10(3)/mL, both in buffer and milk. LM detection was demonstrated using a novel asymmetrically anchored cantilever sensor and a commercially available antibody. Sensor responses were confirmed using a secondary antibody binding step, similar to the sandwich ELISA assays, as a means of signal amplification that also reduced the occurrence of false negatives. Detection of LM at a concentrations of 10(2)/mL was achieved, by incorporating a third antibody binding step, which is an order of magnitude smaller than the infection dose (<1000 cells) for LM. The commercially available antibody for LM used in this work is shown to have low avidity which partially explains the relatively low sensitivity reported for LM as compared to other pathogens.

Rapid and sensitive detection of Giardia lamblia using a piezoelectric cantilever biosensor in finished and source waters.

The current method for detecting the waterborne parasite Giardia lamblia is tedious and requires a preconcentration step. We show for the first time a piezoelectric-excited millimeter-sized cantilever (PEMC) biosensor immobilized with a monoclonal antibody against G. lamblia that exhibits selective and sensitive detection of G. lamblia cysts in several water matrixes (buffer, tap, and river water) at a detection limit of 1-10 cysts/mL without a preconcentration step. The PEMC sensor is a resonance-based device that functions at a high-order mode near 1 MHz. The antibody-immobilized sensor was exposed to 1-10,000 G. lamblia cysts/mL samples in a flow arrangement. When the cysts bind to the antibody on the sensor, the resonant frequency of the cantilever sensor decreases and is recorded continuously. Positive confirmation of sensor detection responses was obtained by environmental scanning electron microscope of sensor surface after detection experiments. Higher sample flow rates (0.5-5.0 mL/min) gave higher sensor detection response. Detecting as few as 10 cysts per mL was achieved in all three water matrixes tested, and significant sensor response was obtained in 15 min. We also show the feasibility of analyzing at a low concentration of 1 cyst/mL in a one liter sample at a high flow rate of 5 mL/min.

10-minute assay for detecting Escherichia coli O157:H7 in ground beef samples using piezoelectric-excited millimeter-size cantilever sensors

We detected Escherichia coli O157:H7 (EC) at approximately 10 cells per ml in spiked ground beef samples in 10 min using piezoelectric-excited millimeter-size cantilever (PEMC) sensors. The composite PEMC sensors have a sensing area of 2 mm2 and are prepared by immobilizing a polyclonal antibody specific to EC on the sensing surface. Ground beef (2.5 g) was spiked with EC at 10 to 10,000 cells per ml in phosphate-buffered saline (PBS). One milliliter of supernatant was removed from the blended samples and used to perform the detection experiments. The total resonant frequency change obtained for the inoculated samples was 138 +/- 9, 735 +/- 23, 2,603 +/- 51, and 7,184 +/- 606 Hz, corresponding to EC concentrations of 10, 100, 1,000, and 10,000 cells per ml, respectively. EC was detected in the sample solution within the first 10 min. The responses of the sensor to positive, negative, and buffer controls were 36 +/- 6, 27 +/- 2, and 2 +/- 7 Hz, respectively. Verification of EC attachment was confirmed by low-pH buffer release (PBS-HCl, pH 2.2), microscopy, and second antibody EC binding postdetection. The results indicate that PEMC sensors can reliably detect EC at less than 10 cells per ml in 10 min without sample preparation and with label-free reagents.

Half Antibody Fragments Improve Biosensor Sensitivity without Loss of Selectivity

We show for the first time that half antibody fragments obtained by reduction via tris(2-carboxyethyl)phosphine (TCEP) gave a larger response with shear-mode resonating mass sensors than physisorbed whole antibody or antibody immobilized via Protein G. The reduced antibody is shown to preserve the antigen-binding region and was determined via the antigen binding response. Reduction exposed the native thiol group in the antibody that readily chemisorbed onto the gold-coated sensor surfaces with the right orientation for antigen binding. Comparing responses obtained on a quartz crystal microbalance for the detection of pathogenic Escherichia coli O157:H7 using TCEP-reduced antibody with native antibody showed that the proposed method enhances device sensitivity. Examining the half antibody fragments for detection of the pathogen in the presence of the nonpathogenic wild strain showed that the antibody fragments retained their specific antigen binding capability without loss of selectivity.

hlyA gene-based sensitive detection of Listeria monocytogenes using a novel cantilever sensor.

Piezoelectric cantilever sensors are shown to exhibit sensitive and selective detection based on an identifying gene from genomic extract at ~10(2)-10(3) cells of foodborne pathogen, Listeria monocytogenes (LM). The study consists of two parts: tests with synthetic genes and experiments starting with whole LM cells. A probe designed for the virulence hemolysin gene, hlyA, was immobilized on the gold-coated sensor, and hybridization detection of a synthetic target (based on hlyA) is shown to span over 6 decades in concentration. Hybridization response was confirmed using two methods: (1) the use of a fluorescent indicator for the presence of double-stranded DNA (ds-DNA) and (2) hybridization response of a secondary single-strand DNA (ss-DNA) to the unhybridized part of the target much like in the enzyme linked immunosorbent assay (ELISA) sandwich format. Hybridization of the secondary ss-DNA tagged to gold nanoparticles amplified as well as confirmed the target hybridization to the hlyA probe on the sensor. Genomic DNA from LM was extracted, sheared, and melted and was exposed to the hlyA probe on the sensor in proteinous background with and without the presence of up to 10(4) times excess nontarget genomic DNA extracted from E.coli JM 101 (EC), for the gene-specific detection of LM. Discernible detection limit of 7 × 10(2) LM cells (equivalent genomic DNA; 2.32 pg) was achieved in proteinous background. The detection limit deteriorated to 7 × 10(3) LM (23 pg of gDNA) in the presence of genomic DNA from EC. Hybridization response times were within ~90 min, thus significantly improving over the conventional detection techniques in detection time at comparable detection limit.