Acoustofluidic particle trapping, manipulation, and release using dynamic-mode cantilever sensors.

We show here that dynamic-mode cantilever sensors enable acoustofluidic fluid mixing and trapping of suspended particles as well as the rapid manipulation and release of trapped micro-particles via mode switching in liquid. Resonant modes of piezoelectric cantilever sensors over the 0 to 8 MHz frequency range are investigated. Sensor impedance response, flow visualization studies using dye and micro-particle tracers (100 µm diameter), and finite element simulations of cantilever modal mechanics and acoustic streaming show fluid mixing and particle trapping configurations depend on the resonant mode shape. We found trapped particles could be: (1) rapidly manipulated on millimeter length scales, and (2) released from the cantilever surface after trapping by switching between low- and high-order resonant modes (less than 250 kHz and greater than 1 MHz, respectively). Such results suggest a potentially promising future for dynamic-mode cantilevers in separations, pumping and mixing applications as well as acoustofluidic-enhanced sensing applications.

Biosensor-based microRNA detection: techniques, design, performance, and challenges.

The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.

Reduction of nonspecific protein adsorption on cantilever biosensors caused by transverse resonant mode vibration.

We examine if vibration of millimeter-sized cantilever sensors can release nonspecifically adsorbed proteins. Integrated electrochemical and mass-change measurement as well as fluorescence assays showed transverse surface vibration released nonspecifically bound proteins in samples prepared at 0.2-3.6 mg bovine serum albumin (BSA) per mL. Extent of release was directly related to magnitude of excitation voltage (Vex) applied to the self-actuating lead zirconate titanate (PZT) cantilever over three log units (0, 10 mV, 100 mV, and 1 V). Vibration-induced release was not instantaneous, but had an apparent first-order rate constant (kapp) which ranged from 0.02-0.1 min(-1). Results suggest significant serum albumin protein release could be achieved using excitation voltages of 1 V in millimeter-sized cantilever sensors. Complementary experiments with thiolated DNA, which binds to surface gold 〈111〉 sites with ∼ four times higher binding energy than BSA, showed negligible release under the same vibration magnitude. The results of the study suggest a direct correlation between surface-adsorbate binding energy and the effectiveness of vibration-induced release. We suggest that the release mechanism includes contributions from surface strain energy, body force, and acoustic streaming-associated hydrodynamic effects. The primary contribution of this study suggests that surface vibration of cantilever sensors may be useful in reducing nonspecific adsorption, especially for biosensing of analytes present in a complex background.

Electrochemical piezoelectric-excited millimeter-sized cantilever (ePEMC) for simultaneous dual transduction biosensing.

A dual mode electrochemical piezoelectric-excited millimeter cantilever (ePEMC) sensor is reported for simultaneous in-liquid biochemical sensing. The ePEMC incorporates mass-sensing measurement of dynamic-mode cantilevers with electrochemical impedance spectroscopy (EIS) commonly employed for transduction in sensitive electrochemical biosensors. Such an integrated design allows for simultaneous and continuous measurement of resonant frequency shift (Δf) and charge transfer resistance (RCT) as a target analyte binds to the sensor gold surface (0.5 mm(2)) via electromechanical and electrochemical impedance spectroscopy, respectively. The properties of ePEMC are demonstrated in three experiments: (1) resonant frequency response to electrochemically-deposited metal thin-films, (2) resonant frequency response to adsorption of thiolated ssDNA and model proteins with subsequent EIS sensing, and (3) simultaneous resonant frequency and charge transfer resistance response to model chemisorption of a short-chain thiol molecule, mercaptohexanol. Adsorption of all model binding analytes caused decrease in sensor resonant frequency and increase in charge transfer resistance. Comparison of sensor response to binding of protein and thiol molecules showed the two simultaneously transduced signals were proportional and showed the same kinetics.

A cantilever biosensor-based assay for toxin-producing cyanobacteria Microcystis aeruginosa using 16S rRNA.

Monitoring of cyanotoxins in source waters is currently done through toxin-targeting assays which suffer from low sensitivity due to poor antibody avidity. We present a biosensor-based method as an alternative for detecting toxin-producing cyanobacteria M. aeruginosa via species-selective region of 16S rRNA at concentrations as low as 50 cells/mL, and over a five-log dynamic range. The cantilever biosensor was immobilized with a 27-base DNA strand that is complementary to the target variable region of 16S rRNA of M. aeruginosa. The cantilever sensor detects mass-changes through shifts in its resonant frequency. Increase in the biosensor's effective mass, caused by hybridization of target strand with the biosensor-immobilized complementary strand, showed consistent and proportional frequency shift to M. aeruginosa concentrations. The sensor hybridization response was verified in situ by two techniques: (a) presence of duplex DNA structure postdetection via fluorescence measurements, and (b) secondary hybridization of nanogold-labeled DNA strands to the captured 16S rRNA strands. The biosensor-based assay, conducted in a flow format (∼ 0.5 mL/min), is relatively short, and requires a postextraction analysis time of less than two hours. The two-step detection protocol (primary and secondary hybridization) is less prone to false negatives, and the technique as a whole can potentially provide an early warning for toxin presence in source waters.

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.

A method for DNA-based detection of E. coli O157:H7 in a proteinous background using piezoelectric-excited cantilever sensors.

We show for the first time the detection of ~700 E. coli O157:H7 (EC) in both buffer and proteinous sample (beef wash) using the virulent gene stx2 as a sensing molecule immobilized on piezoelectric-excited millimeter cantilever (PEMC) sensors without a sample preparation step. Genomic DNA of EC suspended in buffer or beef wash was extracted using a 30-minute procedure, or using a commercial extraction kit, and then was exposed to the sensor immobilized with a 19-mer probe that is complement to stx2 gene. As hybridization occurred, the resonant frequency of the cantilever sensor decreased due to the increased attached mass indicating the presence of stx2 gene in the sample. Wild strain JM101 subjected to the same preparation and procedure did not induce a hybridization response, nor did the genomic extract of EC with a bare sensor. PEMC sensor responses to diluted genomic extracts indicate that a much lower concentration of 700 cells is detectable. In order to compare the results with antibody-based detection, samples with EC at 2500 cells per mL were exposed to antibody-immobilized PEMC sensors which gave reproducible responses confirming previous experiments with such samples.

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.

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.

Torsional and lateral resonant modes of cantilevers as biosensors: alternatives to bending modes.

We show here novel cantilever designs that express torsional and lateral modes exhibit excellent mass-change sensitivity to molecular self-assembly on gold (75-135 fg/Hz) which is superior to that of widely investigated bending modes. Lead zirconate titanate (PZT) millimeter-sized cantilevers were designed with two types of anchor asymmetry that induced expression of either torsional or lateral modes in the 0-80 kHz frequency range. Experiments and supporting calculations show that anchor asymmetry enables resonant mode impedance-coupling. The sensitive torsional and lateral modes enabled measurement of self-assembled monolayer formation rate at picomolar levels. The anchor design principle was extended to microcantilevers via finite element simulations, which caused both 97% sensitivity improvement relative to conventional designs, as well as new nonclassical resonant mode shapes.

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.

pH effect on protein G orientation on gold surfaces and characterization of adsorption thermodynamics.

The pH effect on adsorbed antibody-binding protein (protein G) orientation on gold (Au) and its adsorption thermodynamic characteristics were investigated using quartz crystal microbalance (QCM) and X-ray photoelectron spectroscopy (XPS). The adsorbed protein G orientation was measured by binding response of two antibody-antigen systems: the model bovine serum albumin (BSA) and the foodborne pathogen E. coli O157:H7. Surface coverage was not significantly affected by pH, but its orientation was. The most properly oriented protein G for antibody binding was achieved at near-neutral pH. Adsorption was verified by XPS measurements using nitrogen (N) 1s, oxygen (O) 1s, and Au 4p peak heights. Adsorption energetics were determined by van't Hoff and Langmuir kinetic analyses of adsorption data obtained at 296, 303, and 308 K. Large characteristic entropy change of protein adsorption was observed (ΔS° = 0.52 ± 0.01 kcal/mol·K). The adsorption process was not classical physisorption but exhibited chemisorption characteristics based on significant enthalpy change (ΔH° = -25 ± 6 kcal/mol).

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.

Cell viability measurement using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester and a cantilever sensor.

Detection of viable pathogenic bacteria has widespread application in food safety and human health. Antibody-based methods require a growth step which limits time-to-results performance. In this study, we use a mass-change sensitive cantilever biosensor and a probe, 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM), that accumulates only in live cells inducing a mass-change response to determine the cell viability in a short time. A poly-L-lysine coated sensor immobilized with live Escherichia coli JM101 (a surrogate for a pathogenic target) at various concentrations was exposed to BCECF-AM in a flow arrangement. A larger resonant frequency decrease in response to 100 µL of 60 µM BCECF-AM was observed when the sensor surface cell concentration was increased from 1 090 ± 580 to 3 960 ± 370 cells/mm(2) (n = 5). A log-linear relationship between the sensor surface cell concentration and frequency response was obtained in the range of 1 000-4 000 cells/mm(2) and as low as ∼2 000 viable E. coli cells were rapidly detected (<1 h).

Highly sensitive and rapid detection of microcystin-LR in source and finished water samples using cantilever sensors.

Microcystin-leucine-arginine (MCLR) is one of the toxic microcystin congeners produced by the common cyanobacteria, blue-green algae. A piezoelectric-excited millimeter-sized cantilever (PEMC) sensor was developed for the sensitive detection of MCLR in a flow format using both monoclonal and polyclonal antibodies that bind specifically to MCLR. PEMC is a resonant cantilever sensor whose resonant frequency decreases as target analyte binds to its surface. Monoclonal antibody against MCLR was immobilized on the sensor surface via amine coupling. As the toxin in the sample water bound to the antibody, resonant frequency decreased proportional to toxin concentration. Three water matrices, namely buffer, tap water, and river water, were spiked with MCLR standards and were successfully detected in the dynamic range of 1 pg/mL to 100 ng/mL (effective concentration -250 fg/mL to 25 ng/mL). The sensor response was characterized by a log-linear relationship between resonant frequency change and MCLR concentration. Positive verification of MCLR detection was confirmed by a sandwich binding on the sensor with a second antibody binding to MCLR on the sensor (attached in first detection step) which caused a further resonant frequency decrease. We show for the first time that MCLR in various water samples can be detected at 1 pg/mL.

Detection of Cryptosporidium parvum in buffer and in complex matrix using PEMC sensors at 5 oocysts mL(-1).

We show for the first time that a piezoelectric-excited millimeter-sized cantilever (PEMC) sensor can detect five Cryptosporidium parvum oocysts in 25% milk in PBS background in a flow format (1 mL min(-1)). To improve sensitivity, a secondary antibody (murine IgM) was used to confirm the attachment of oocysts to the sensor. PEMC sensor is a resonant-mode cantilever sensor whose resonant frequency decreases when target analyte binds to its surface. The sensor was functionalized with Protein G, and then immobilized with goat polyclonal IgG anti-C. parvum for oocysts detection. In the dynamic range of 50-10,000 oocysts mL(-1) the sensor response is characterized by a semi-log relationship between resonant frequency response and C. parvum oocysts concentration. In 25% milk background, binding kinetics was slower and total sensor response was lower (approximately 45%) than in water-like buffer.

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.

Measurement and modeling of diffusion kinetics of a lipophilic molecule across rabbit cornea.

PURPOSE: To develop a kinetic model for representing the diffusion and partitioning of Rhodamine B (RhB), a fluorescent lipophilic molecule, across the cornea for gaining insights into pharmacokinetics of topical drugs to the eye. METHODS: Rabbit corneas mounted underneath a custom-built scanning microfluorometer were perfused with Ringers on both sides of the tissue. After a step change in RhB on the tear side, transients of trans-corneal fluorescence of RhB were measured at a depth resolution approximately 8 microm. RESULTS: RhB distribution exhibited discontinuities at the interface between epithelium and stroma, and between stroma and endothelium. In each of the layers, fluorescence was non-uniform. Fluorescence was elevated in the epithelium and endothelium relative to the stroma. Modeling of RhB transport by diffusion in each layer and stipulation of partitioning of RhB at the cellular interfaces were required to account for trans-corneal penetration kinetics of RhB. The model parameters, estimated using the unsteady state trans-corneal RhB profiles, were found to be sensitive, and the model predicted the experimental profiles accurately. CONCLUSIONS: Conventional pharmacokinetic models that depict cornea as a single compartment do not predict the depth-dependent kinetics of RhB penetration. The proposed model incorporates realistic transport mechanisms and thereby highlights the influence of physicochemical properties of drugs on trans-corneal kinetics.

Expression of picogram sensitive bending modes in piezoelectric cantilever sensors with nonuniform electric fields generated by asymmetric electrodes.

Single-layer uniform cross-sectioned piezoelectric macro-cantilevers fabricated with an asymmetric electrode configuration enabled electrical measurement of picogram-sensitive resonant bending modes in liquids. Bending modes were otherwise not electrically measurable without excitation by a nonuniform electric field created by the geometric asymmetry in electrode design used. Electrode modification was confirmed by energy-dispersive X-ray spectroscopy (EDS). Mass-change sensitivity was tested using both bulk density changes and surface chemisorption experiments in a continuous flow apparatus. Significant response to density changes as small as 0.004 g/mL was measured. A sensitivity limit of ∼1 picogram in liquid was determined from 1-dodecanethiol chemisorption experiments. The sensitivity decreased with chemisorbed mass and was log-linear over five orders of magnitude. The observed resonance responses were in agreement with previously reported models of resonating cantilever sensors. This work demonstrates experimentally for the first time that introducing electrode asymmetry enables measurement of bending modes in cantilevers containing only a single piezoelectric layer.

Sensitive and selective detection of mycoplasma in cell culture samples using cantilever sensors.

In this article we report a new biosensor-based method that is more sensitive and rapid than the current approach for detecting mycoplasma in cell culture samples. Piezoelectric-excited millimeter-sized cantilever (PEMC) sensors respond to mass change via resonant frequency change. They are sensitive at femtogram level and can be used directly in liquid for label-free detection. Common cell culture contaminant, Acholeplasma laidlawii was detected in both buffer and cell culture medium. Two different sources (positive control from a commercial kit and ATCC 23206) were analyzed using antibody-immobilized PEMC sensor. Resonant frequency decrease caused by binding of A. laidlawii was monitored in real-time using an impedance analyzer. Positive detection was confirmed by a second antibody binding. The limit of detection (LOD) was lower than 10(3) CFU/mL in cell culture medium using PEMC sensor while parallel ELISA assays showed LOD as 10(7) CFU/mL. This study shows that PEMC sensor can be used for sensitive and rapid mycoplasma detection in cell culture samples.