Tunable patterning of microparticles and cells using standing surface acoustic waves

We have developed an acoustic-based tunable patterning technique by which microparticles or cells can be arranged into reconfigurable patterns in microfluidic channels. In our approach, we use pairs of slanted-finger interdigital transducers (SFITs) to generate a tunable standing surface acoustic wave field, which in turn patterns microparticles or cells in one- or two-dimensional arrays inside the microfluidic channels--all without the assistance of fluidic flow. By tuning the frequency of the input signal applied to the SFITs, we have shown that the cell pattern can be controlled with tunability of up to 72%. This acoustic-based tunable patterning technique has the advantages of wide tunability, non-invasiveness, and ease of integration to lab-on-a-chip systems, and shall be valuable in many biological and colloidal studies.     

A cell-based biosensor for real-time detection of cardiotoxicity using lensfree imaging

A portable and cost-effective real-time cardiotoxicity biosensor was developed using a CMOS imaging module extracted from a commercially available webcam. The detection system consists of a CMOS imaging module, a white LED and a pinhole. Real-time image processing was conducted by comparing reference and live frame images. To evaluate the engineered system, the effects of two different drugs, isoprenaline and doxorubicin, on the beating rate and beat-to-beat variations of ESC-derived cardiomyocytes were measured. The detection system was used to conclude that the beat-to-beat variability increased under treatment with both isoprenaline and doxorubicin. However, the beating rates increased upon the addition of isoprenaline but decreased for cultures supplemented with doxorubicin. Moreover, the response time for both the beating rates and the beat-to-beat variability of ESC-derived cardiomyocytes under treatment of isoprenaline was shorter than for doxorubicin, although the amount of isoprenaline used in the measurement was three orders of magnitude lower than that of doxorubicin. Given its ability to perform real-time cell monitoring in a simple and inexpensive manner, the proposed system may be useful for a range of cell-based biosensing applications.     

Uniform mixing in paper-based microfluidic systems using surface acoustic waves

Paper-based microfluidics has recently received considerable interest due to their ease and low cost, making them extremely attractive as point-of-care diagnostic devices. The incorporation of basic fluid actuation and manipulation schemes on paper substrates, however, afford the possibility to extend the functionality of this simple technology to a much wider range of typical lab-on-a-chip operations, given its considerable advantages in terms of cost, size and integrability over conventional microfluidic substrates. We present a convective actuation mechanism in a simple paper-based microfluidic device using surface acoustic waves to drive mixing. Employing a Y-channel structure patterned onto paper, the mixing induced by the 30 MHz acoustic waves is shown to be consistent and rapid, overcoming several limitations associated with its capillary-driven passive mixing counterpart wherein irreproducibilities and nonuniformities are often encountered in the mixing along the channel--capillary-driven passive mixing offers only poor control, is strongly dependent on the paper's texture and fibre alignment, and permits backflow, all due to the scale of the fibres being significant in comparison to the length scales of the features in a microfluidic system. Using a novel hue-based colourimetric technique, the mixing speed and efficiency is compared between the two methods, and used to assess the effects of changing the input power, channel tortuousity and fibre/flow alignment for the acoustically-driven mixing. The hue-based technique offers several advantages over grayscale pixel intensity analysis techniques in facilitating quantification without limitations on the colour contrast of the samples, and can be used, for example, for quantification in on-chip immunochromatographic assays.     

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC₅₀) values of the SPR assay were 28.8 and 14.9 ng mL⁻¹ for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg⁻¹ for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg⁻¹ for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.     

Separation of platelets from whole blood using standing surface acoustic waves in a microchannel

Platelet separation from blood is essential for biochemical analyses and clinical diagnosis. In this article, we propose a method to separate platelets from undiluted whole blood using standing surface acoustic waves (SSAWs) in a microfluidic device. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with interdigitated transducer (IDT) electrodes patterned on a piezoelectric substrate. To avoid shear-induced activation of platelets, the blood sample flow was hydrodynamically focused by introducing sheath flow from two side-inlets and pressure nodes were designed to locate at side walls. By means of flow cytometric analysis, the RBC clearance ratio from whole blood was found to be over 99% and the purity of platelets was close to 98%. Conclusively, the present technique using SSAWs can directly separate platelets from undiluted whole blood with higher purity than other methods.     

Focusing surface acoustic waves assisted electrochemical detector in microfluidics

This article demonstrates a novel electrochemical detection device. The device is composed by two focusing interdigital transducers for exciting focused surface acoustic waves by applying an AC signal, a three-electrode system for electrochemical measurement, and a liquid pool for holding liquid on a LiNbO₃ wafer. The amperometry current of ferrocenecarboxylic acid and potassium phosphate buffer solution is used to characterize the detection sensitivity. Two experiments are carried out to optimize the device design. The result shows that the two focusing interdigital transducers with arc degree 30° and distance 5 mm can remarkably enhance the liquid mixing rate. Under this condition, the oxidation current is about 27 times larger than that without surface acoustic wave stirring.     

Gold nanoparticle-based immunoassay by using non-stripping chemiluminescence detection

A novel microplate-compatible chemiluminescence (CL) immunoassay has been developed for the determination of human immunoglobulin G (IgG) based on the luminol-AgNO(3)-gold nanoparticles CL system. Polystyrene microtiter plates were used for both immunoreactions and CL measurements. The primary antibody, goat-anti-human IgG, was first immobilized on polystyrene microwells. Then the antigen (human IgG) and the gold-labeled second antibody were connected to the microwells successively to form a sandwich-type immunocomplex. The gold label could trigger the reaction between luminol and AgNO(3), accompanied by light emission. Under the optimized conditions, the CL intensity of the system was linear with the logarithm of the concentration of human IgG in the range from 25 to 5000 ng mL(-1), with a detection limit of 12.8 ng mL(-1) ( approximately 80 pM) at a signal to noise ratio of three (S/N = 3). Compared with other reported CL immunoassay method based on gold labels, the proposed CL protocol avoids a strict stripping procedure or difficult to control synthesis processes, making the method more simple, time-saving and easily automated. The present CL method is promising for the determination of clinically important bioactive analytes.     

Interaction-free, automatic, on-chip fluid routing by surface acoustic waves

By exploiting the resonant coupling between a travelling wave and the stationary modes of a cavity, we present the first scheme for integrated automatic interaction-free surface acoustic wave routing of fluids. Our scheme opens the way to the implementation of logic gates based on instantaneous liquid distribution.     

Enzyme-amplified protein microarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

Two immunoassay platforms were developed for either the sensitive or rapid detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. These antibodies also bind the same epitopes of the receptor binding domain present on a nontoxic recombinant heavy chain fragment used for assay development and testing in the current study. An enzyme-linked immunosorbent assay (ELISA) microarray using tyramide amplification for localized labeling was developed for the specific and sensitive detection of BoNT. This assay has the sensitivity to detect BoNT in buffer and blood plasma samples down to 14 fM (1.4 pg mL⁻¹). Three capture antibodies and one antibody combination were compared in the development of this assay. Using a selected pair from the same set of recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days. The renewable surface assay is less sensitive but much faster, providing results in less than 10 min.     

Real-time immunoassay of ferritin using surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used for the first time to determine the concentration of ferritin in both HBS-EP buffer and serum. The monoclonal antibody was immobilized on the carboxymethyl dextran-modified gold surface by an amine coupling method. The interaction of antibody with antigen was monitored in real-time. The signal was enhanced by sandwich amplification strategy to improve the sensitivity and specificity of the immunoassay, especially in serum. The linear range of the assay in serum is over 30-200 ng ml⁻¹ with the detection limit of 28 ng ml⁻¹. The sensitivity, specificity, and reproducibility of the assay are satisfactory. The analyte and enhancement antibody-binding surface could be regenerated by pH 2.0 glycine-HCl buffer and the same antibody-immobilized surface could be used for more than 50 cycles of ferritin binding and regeneration.     

DNA concentration modulation on supported lipid bilayers switched by surface acoustic waves

Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays; however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (~10 μm). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion.     

Rapid detection of Mycobacterium tuberculosis cells by using microtip-based immunoassay

This paper describes a microtip-based approach of concentrating target analytes for a highly sensitive bioassay. As an example, rapid screening of bacterial whole cells is presented to detect Mycobacterium tuberculosis (MTB), a pathogenic bacterium for human tuberculosis (TB). The concentration and detection is performed with three sequential steps of (1) attracting bacterial whole cells in the vicinity of a microtip by alternating current electroosmotic flow; (2) capturing the cells onto the microtip by capillary action; (3) binding fluorophore-labeled polyclonal antibodies to the cells followed by fluorescence measurement (immunofluorescence). Through this mechanism, MTB cells have been detected to the concentration of 8,000 cells/mL within 10 min. This sensitivity is comparable to that of Ziehl–Neelsen smear microscopy, a common culture-free screening method for diagnosis of TB. For comparison, Escherichia coli O157:H7 cells have also been detected to the concentration of 30,000 cells/mL in the same way.     

Tritoroidal particle rings formation in open microfluidics induced by standing surface acoustic waves

In this paper, the particle movements in a sessile droplet induced by standing surface acoustic waves (SSAWs) are studied. Tritoroidal particle rings are formed under the interaction of acoustic field and electric field. The experimental results demonstrate that the electric field plays an important role in patterning nanoparticles. The electric field can define the droplet shape due to electrowetting. When the droplet approximates a hemisphere, the acoustic radiation force induced by SSAWs drives the particles to form tritoroidal particle rings. When the droplet approximates a convex plate, the drag force induced by acoustic steaming drives the particle to move. The results will be useful for better understanding the nanoparticle movements in a sessile droplet, which is important to explain the mechanism that SSAWs enhance reaction and crystallization in droplet.     

Recent advances in particle and droplet manipulation for lab-on-a-chip devices based on surface acoustic waves

Manipulation of microscale particles and fluid liquid droplets is an important task for lab-on-a-chip devices for numerous biological researches and applications, such as cell detection and tissue engineering. Particle manipulation techniques based on surface acoustic waves (SAWs) appear effective for lab-on-a-chip devices because they are non-invasive, compatible with soft lithography micromachining, have high energy density, and work for nearly any type of microscale particles. Here we review the most recent research and development of the past two years in SAW based particle and liquid droplet manipulation for lab-on-a-chip devices including particle focusing and separation, particle alignment and patterning, particle directing, and liquid droplet delivery.     

Competitive immunoassay for vancomycin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay using capillary electrophoresis with laser-induced fluorescence was developed for vancomycin. Capillary electrophoresis using a Tris-glycine running buffer provided adequate separation of the antibody-bound from the unbound fluorescent probe (tracer) in less than 4 min. Laser-induced fluorescence polarization (LIFP) provided high sensitivity detection and simultaneous monitoring of fluorescence intensity and polarization. A fluorescence polarization value of 0.30 confirmed the formation of the antibody-tracer complex. Calibration curves showed a working linear range of 2-3 orders of magnitude with a minimum detectable concentration of 0.98 ng mL(-1) (or 1.1 fg vancomycin). Clinical samples obtained from patients undergoing vancomycin treatment were analyzed for vancomycin and the results correlated well with a standard immunoassay based on latex particle detection that was routinely used by a hospital laboratory. Only 1/10 of the reagents were needed as compared with the standard immunoassay.     

Focusing microparticles in a microfluidic channel with standing surface acoustic waves (SSAW)

We introduce a novel on-chip microparticle focusing technique using standing surface acoustic waves (SSAW). Our method is simple, fast, dilution-free, and applicable to virtually any type of microparticle.     

Label-free detection of neuron-drug interactions using acoustic and Kelvin vibrational fields

Kelvin and acoustic fields of high-frequency have been employed in the non-invasive investigation of immortalized hypothalamic neurons, in order to assess their response to different concentrations of specific drugs, toxins, a stress-reducing hormone and neurotrophic factors. In an analytical systems biology approach, this work constitutes a first study of living neuron cultures by scanning Kelvin nanoprobe (SKN) and thickness shear mode (TSM) acoustic wave techniques. N-38 hypothalamic mouse neurons were immobilized on the gold electrode of 9 MHz TSM acoustic wave devices and gold-coated slides for study by SKN. The neurons were exposed to the neurochemicals betaseron, forskolin, TCAP, and cerebrolysin. Signals were collected with the TSM in real-time mode, and with the SKN in scanning and real-time modes, as the drugs were applied at biologically significant concentrations. With the TSM, for all drugs, some frequency and resistance shifts were in the same direction, contrary to normal functioning for this type of instrument. Possible mechanisms are presented to explain this behaviour. An oscillatory signal with periodicity of approximately 2 min was observed for some neuron-coated surfaces, where the amplitude of these oscillations was altered upon application of certain neurotrophic factors. These two new techniques present novel and non-invasive electrodeless methods for detecting changes at the cellular level caused by a variety of neuroactive compounds, without killing or destroying the neurons.     

Highly sensitive detection of human cardiac myoglobin using a reverse sandwich immunoassay with a gold nanoparticle-enhanced surface plasmon resonance biosensor

A highly sensitive reverse sandwich immunoassay for the detection of human cardiac myoglobin (cMb) in serum was designed utilizing a gold nanoparticle (AuNP)-enhanced surface plasmon resonance (SPR) biosensor. First, a monoclonal anti-cMb antibody (Mab1) was covalently immobilized on the sensor surface. AuNPs were covalently conjugated to the second monoclonal anti-cMb antibody (Mab2) to form an immuno-gold reagent (Mab2-AuNP). The reverse sandwich immunoassay consists of two steps: (1) mixing the serum sample with Mab2-AuNP and incubation for the formation of cMb/Mab2-AuNP complexes and (2) sample injection over the sensor surface and evaluation of the Mab1/cMb/Mab2-AuNP complex formation, with the subsequent calculation of the cMb concentration in the serum. The biosensor signal was amplified approximately 30-fold compared with the direct reaction of cMb with Mab1 on the sensor surface. The limit of detection of cMb in a human blood serum sample was found to be as low as 10 pM (approx. 0.18 ng mL⁻¹), and the inter-assay coefficient of variation was less than 3%. Thus, the developed SPR-based reverse sandwich immunoassay has a sensitivity that is sufficient to measure cMb across a wide range of normal and pathological concentrations, allowing an adequate estimation of the disease severity and the monitoring of treatment.     

PDMS microfludic device for optical detection of protein immunoassay using gold nanoparticles

A simple but highly specific immunoassay system for goat anti-human IgG has been developed using gold nanoparticles and microfluidic techniques. The assay is based on the deposition of gold nanoparticles that are coated with protein antigens in the presence of their corresponding antibodies to microfluidic channel surface. The effects of time accumulation, the flow velocity, and the concentration of antibodies to the red light absorption percentage (RAP) of deposition were investigated with an ordinary optical microscope. By controlling the reaction time and flow velocity, a dynamic range of 3 orders of magnitude and a detection sensitivity of 10 ng ml(-1) of goat anti-human IgG were achieved. Because of its simplicity and flexibility, this new technique should be useful for fast, highthroughput screening of antibodies in clinical diagnostic applications.     

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.