Microwell device for targeting single cells to electrochemical microelectrodes for high-throughput amperometric detection of quantal exocytosis.

Electrochemical microelectrodes are commonly used to detect spikes of amperometric current that correspond to exocytosis of oxidizable transmitter from individual vesicles, i.e., quantal exocytosis. We are developing transparent multielectrochemical electrode arrays on microchips in order to automate measurement of quantal exocytosis. Here, we report development of an improved device to target individual cells to each microelectrode in an array. Efficient targeting (~75%) is achieved using cell-sized microwell traps fabricated in SU-8 photoresist together with patterning of poly(l-lysine) in register with electrodes to promote cell adhesion. The surface between electrodes is made resistant to cell adhesion using poly(ethylene glycol) in order to facilitate movement of cells to electrode "docking sites". We demonstrate the activity of the electrodes using the test analyte ferricyanide and perform recordings of quantal exocytosis from bovine adrenal chromaffin cells on the device. Multiple cell recordings on a single device demonstrate the consistency of spike measurements, and multiple recordings from the same electrodes demonstrate that the device can be cleaned and reused without degradation of performance. The new device will enable high-throughput studies of quantal exocytosis and may also find application in rapidly screening drugs or toxins for effects on exocytosis.

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND: Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS: In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS: Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.

A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform.

Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-ß-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases ß-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and allowing the procedure to be completed in hours rather than days.

Specific and targeted detection of viable Escherichia coli O157:H7 using a sensitive and reusable impedance biosensor with dose and time response studies.

A gold interdigitated microelectrode (IME) impedance biosensor was fabricated for the detection of viable Escherichia coli O157:H7. This sensor was fabricated using lithography techniques. The surface of the electrode was immobilized with anti-E. coli IgG antibodies. This approach is different from other studies where the change in impedance is measured in terms of growth of bacteria on the electrode, rather then the antibody/antigen bonding. The impedance values were recorded for frequency ranges between 100 Hz and 10 MHz. The working range of the dose response for this device was found to be between 2.5×10(4) CFU ml(-1) and 2.5×10(7) CFU ml(-1). The time response studies indicated that antibody/antigen binding is not a function of time, but can decrease if excess times are allowed for binding. It was observed that the impedance values for 60 min antibody/antigen binding were higher than the impedance values for 120 min binding time. The main advantages of the reported device are that, it provides for both qualitative and quantitative detection in 3h while other impedance sensors reported earlier may take up to 24h for detection. If enrichment steps are required then it may take 3-4 days to infer the results. This sensor can be used to detect different types of bacteria by immobilizing the antigen specific antibody. Most of the sensors are not reusable since they either use enzymes or enrichment steps for detection but this device can be reused, following a cleaning protocol which is easy to follow. Each device was used at least five times. The simplicity of this sensor and the ease of fabrication make this sensor a useful alternate to the microfluidics and enzyme based impedance sensors, which are relatively more difficult to fabricate, need programmable fluidic injection pumps to push the sample through the channel, suffer from limitation of coagulation and are difficult to clean.

Automated targeting of cells to electrochemical electrodes using a surface chemistry approach for the measurement of quantal exocytosis.

Here we describe a method to fabricate a multi-channel high-throughput microchip device for measurement of quantal transmitter release from individual cells. Instead of bringing carbon-fiber electrodes to cells, the device uses a surface chemistry approach to bring cells to an array of electrochemical microelectrodes. The microelectrodes are small and "cytophilic" in order to promote adhesion of a single cell whereas all other areas of the chip are covered with a thin "cytophobic" film to block cell attachement and facilitate movement of cells to electrodes. This cytophobic film also insulates unused areas of the conductive film, thus the alignment of cell docking sites to working electrodes is automatic. Amperometric spikes resulting from single-granule fusion events were recorded on the device and had amplitudes and kinetics similar to those measured using carbon-fiber microelectrodes. Use of this device will increase the pace of basic neuroscience research and may also find applications in drug discovery or validation.

Preferential cell attachment to nitrogen-doped diamond-like carbon (DLC:N) for the measurement of quantal exocytosis.

Electrochemical measurement of transmitter or hormone release from individual cells on microchips has applications both in basic science and drug screening. High-resolution measurement of quantal exocytosis requires the working electrode to be small (cell-sized) and located in immediate proximity to the cell. We examined the ability of candidate electrode materials to promote the attachment of two hormone-secreting cell types as a mechanism for targeting cells for to recording electrodes with high precision. We found that nitrogen-doped diamond-like carbon (DLC:N) promoted cell attachment relative to other materials tested in the rank order of DLC:N>In(2)O(3)/SnO(2) (ITO), Pt>Au. In addition, we found that treating candidate electrode materials with polylysine did not increase attachment of chromaffin cells to DLC:N, but promoted cell attachment to the other tested materials. We found that hormone-secreting cells did not attach readily to Teflon AF as a potential insulating material, and demonstrated that patterning of Teflon AF leads to selective cell targeting to DLC:N "docking sites". These results will guide the design of the next generation of biochips for automated and high-throughput measurement of quantal exocytosis.

A microfluidic cell trap device for automated measurement of quantal catecholamine release from cells.

Neurons and endocrine cells secrete neurotransmitter and hormones in discrete packets in a process called quantal exocytosis. Electrochemical microelectrodes can detect spikes in current resulting from the oxidation of individual quanta of transmitter only if the electrodes are small and directly adjacent to release sites on the cell. Here we report development of a microchip device that uses microfluidic traps to automatically target individual or small groups of cells to small electrochemical electrodes. Microfluidic channels and traps were fabricated by multi-step wet etch of a silicon wafer whereas Pt electrodes were patterned in register with the trap sites. We demonstrate high-resolution amperometric measurement of quantal exocytosis of catecholamines from chromaffin cells on the device. This reusable device is a step towards developing high-throughput lab-on-a-chip instruments for recording quantal exocytosis to increase the pace of basic neuroscience research and to enable screening of drugs that target exocytosis.