On-chip contactless four-electrode conductivity detection for capillary electrophoresis devices

In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 microM for potassium was demonstrated.     

Locally oxidized silicon surface-plasmon Schottky detector for telecom regime

We experimentally demonstrate an on-chip nanoscale silicon surface-plasmon Schottky photodetector based on internal photoemission process and operating at telecom wavelengths. The device is fabricated using a self-aligned approach of local-oxidation of silicon (LOCOS) on silicon on insulator substrate, which provides compatibility with standard complementary metal-oxide semiconductor technology and enables the realization of the photodetector and low-loss bus photonic waveguide at the same fabrication step. Additionally, LOCOS technique allows avoiding lateral misalignment between the silicon surface and the metal layer to form a nanoscale Schottky contact. The fabricated devices showed enhanced detection capability for shorter wavelengths that is attributed to increased probability of the internal photoemission process. We found the responsivity of the nanodetector to be 0.25 and 13.3 mA/W for incident optical wavelengths of 1.55 and 1.31 μm, respectively. The presented device can be integrated with other nanophotonic and nanoplasmonic structures for the realization of monolithic opto-electronic circuitry on-chip.     

An integrated fritless column for on-chip capillary electrochromatography with conventional stationary phases

A new polymer device for use with conventional particulate stationary phases for on-chip, fritless, capillary electrochromatography (CEC) has been realized. The structure includes an injector and a tapered column in which the particles of the stationary phase are retained and stabilized. The chips were easily fabricated in poly(dimethylsiloxane) using deep-reactive-ion-etched silicon masters, and tested using a capillary electrophoretic separation of FITC-labeled amino acids. To perform CEC, the separation channel was packed using a vacuum with 3-microm, octadecylsilanized silica microspheres. The packing was stabilized in the column by a thermal treatment, and its stability and quality were evaluated using in-column indirect fluorescence detection. The effects of voltage on electro-osmotic flow and on efficiency were investigated, and the separation of two neutral compounds was achieved in less than 15 s.     

Enhanced 3D fluorescence live cell imaging on nanoplasmonic substrate

We have created a randomly distributed nanocone substrate on silicon coated with silver for surface-plasmon-enhanced fluorescence detection and 3D cell imaging. Optical characterization of the nanocone substrate showed it can support several plasmonic modes (in the 300-800 nm wavelength range) that can be coupled to a fluorophore on the surface of the substrate, which gives rise to the enhanced fluorescence. Spectral analysis suggests that a nanocone substrate can create more excitons and shorter lifetime in the model fluorophore Rhodamine 6G (R6G) due to plasmon resonance energy transfer from the nanocone substrate to the nearby fluorophore. We observed three-dimensional fluorescence enhancement on our substrate shown from the confocal fluorescence imaging of chinese hamster ovary (CHO) cells grown on the substrate. The fluorescence intensity from the fluorophores bound on the cell membrane was amplified more than 100-fold as compared to that on a glass substrate. We believe that strong scattering within the nanostructured area coupled with random scattering inside the cell resulted in the observed three-dimensional enhancement in fluorescence with higher photostability on the substrate surface.     

Integrated hydrogenated amorphous Si photodiode detector for microfluidic bioanalytical devices

Hydrogenated amorphous silicon (a-Si:H) PIN photodiodes have been developed and characterized as fluorescence detectors for microfluidic analysis devices. A discrete a-Si:H photodiode is first fabricated on a glass substrate and used to detect fluorescent dye standards using conventional confocal microscopy. In this format, the limit of detection for fluorescein flowing in a 50-microm deep channel is 680 pM (S/N = 3). A hybrid integrated detection system consisting of a half-ball lens, a ZnS/YF3 multilayer optical interference filter with a pinhole, and an annular a-Si:H photodiode is also developed that allows the laser excitation to pass up through the central aperture in the detector. Using this integrated detection device, the limit of detection for fluorescein is 17 nM, and DNA fragment sizing and chiral analysis of glutamic acid are successfully performed. The a-Si:H detector exhibits high sensitivity at the emission wavelengths of commonly used fluorescent dyes and is readily microfabricated and integrated at low cost making it ideal for portable microfluidic bioanalyzers and emerging large scale integrated microfluidic technologies.     

Electrophoretic separation of proteins on a microchip with noncovalent, postcolumn labeling

Proteins were separated by microchip capillary electrophoresis and labeled on-chip by postcolumn addition of a fluorogenic dye, NanoOrange, for detection by laser-induced fluorescence. NanoOrange binds noncovalently with hydrophobic protein regions to form highly fluorescent complexes. Kinetic measurements of complex formation on the microchips suggest that the reaction rate is near the diffusion limit under the conditions used for protein separation. Little or no band broadening is caused by the postcolumn labeling step. Lower limits of detection for model proteins, alpha-lactalbumin, beta-lactoglobulin A, and beta-lactoglobulin B, were <0.5 pg (approximately 30 amol) of injected sample. The relative fluorescence and reaction rates are compared with those of a number of other fluorogenic dyes used for protein labeling.     

High-quality substrate for fluorescence enhancement using agarose-coated silica opal film

To improve the sensitivity of fluorescence detection in biochip, a new kind of substrates was developed by agarose coating on silica opal film. In this study, silica opal film was fabricated on glass substrate using the vertical deposition technique. It can provide stronger fluorescence signals and thus improve the detection sensitivity. After coating with agarose, the hybrid film could provide a 3D support for immobilizing sample. Comparing with agarose-coated glass substrate, the agarose-coated opal substrates could selectively enhance particular fluorescence signals with high sensitivity when the stop band of the silica opal film in the agarose-coated opal substrate overlapped the fluorescence emission wavelength. A DNA hybridization experiment demonstrated that fluorescence intensity of special type of agarose-coated opal substrates was about four times that of agarose-coated glass substrate. These results indicate that the optimized agarose-coated opal substrate can be used for improving the sensitivity of fluorescence detection with high quality and selectivity.     

Spatially resolved non-invasive chemical stimulation for modulation of signalling in reconstructed neuronal networks.

Functional coupling of reconstructed neuronal networks with microelectronic circuits has potential for the development of bioelectronic devices, pharmacological assays and medical engineering. Modulation of the signal processing properties of on-chip reconstructed neuronal networks is an important aspect in such applications. It may be achieved by controlling the biochemical environment, preferably with cellular resolution. In this work, we attempt to design cell-cell and cell-medium interactions in confined geometries with the aim to manipulate non-invasively the activity pattern of an individual neuron in neuronal networks for long-term modulation. Therefore, we have developed a biohybrid system in which neuronal networks are reconstructed on microstructured silicon chips and interfaced to a microfluidic system. A high degree of geometrical control over the network architecture and alignment of the network with the substrate features has been achieved by means of aligned microcontact printing. Localized non-invasive on-chip chemical stimulation of micropatterned rat cortical neurons within a network has been demonstrated with an excitatory neurotransmitter glutamate. Our system will be useful for the investigation of the influence of localized chemical gradients on network formation and long-term modulation.     

Fluorophore-Decorated Mie Resonant Silicon Nanosphere for Scattering/Fluorescence Dual-Mode Imaging

Inorganic nanoparticles with multiple functions have been attracting attention as multimodal nanoprobes in bioimaging, biomolecule detection, and medical diagnosis and treatment. A drawback of conventional metallic nanoparticle-based nanoprobes is the Ohmic losses that lead to fluorescence quenching of attached molecules and local heating under light irradiation. Here, metal-free nanoprobes capable of scattering/fluorescence dual-mode imaging are developed. The nanoprobes are composed of a silicon nanosphere core having efficient Mie scattering in the visible to near infrared range and a fluorophore doped silica shell. The dark-field scattering and photoluminescence images/spectra for nanoprobes made from different size silicon nanospheres and different kinds of fluorophores are studied by single particle spectroscopy. The fluorescence spectra are strongly modified by the Mie modes of a silicon nanosphere core. By comparing scattering and fluorescence spectra and calculated Purcell factors, the fluorescence enhancement factor is quantitatively discussed. In vitro scattering/fluorescence imaging studies on human cancer cells demonstrate that the developed nanoparticles work as scattering/fluorescence dual-mode imaging nanoprobes.     

Detection of 100 aM fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis

We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.     

On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets

The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 106 smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet motion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of approximately 18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.     

Inkjet printed surface enhanced Raman spectroscopy array on cellulose paper

A novel, ultra low-cost surface enhanced Raman spectroscopy (SERS) substrate has been developed by modifying the surface chemistry of cellulose paper and patterning nanoparticle arrays, all with a consumer inkjet printer. Micro/nanofabrication of SERS substrates for on-chip chemical and biomolecular analysis has been under intense investigation. However, the high cost of producing these substrates and the limited shelf life severely limit their use, especially for routine laboratory analysis and for point-of-sample analysis in the field. Paper-based microfluidic biosensing systems have shown great potential as low-cost disposable analysis tools. In this work, this concept is extended to SERS-based detection. Using an inexpensive consumer inkjet printer, cellulose paper substrates are modified to be hydrophobic in the sensing regions. Synthesized silver nanoparticles are printed onto this hydrophobic paper substrate with microscale precision to form sensing arrays. The hydrophobic surface prevents the aqueous sample from spreading throughout the paper and thus concentrates the analyte within the sensing region. A SERS fingerprint signal for Rhodamine 6G dye was observed for samples with as low as 10 femtomoles of analyte in a total sample volume of 1 μL. This extraordinarily simple technique can be used to construct SERS microarrays immediately before sample analysis, enabling ultra low-cost chemical and biomolecular detection in the lab as well as in the field at the point of sample collection.     

Serial immunoassays in parallel on a microfluidic chip for monitoring hormone secretion from living cells

A microfluidic chip that allows for the continuous monitoring of cellular secretions from multiple independent living samples was developed. Performance of the device was characterized through the analysis of insulin secretion from islets of Langerhans. The chip contained four individual channel networks, each capable of performing electrophoresis-based immunoassays of the perfusate from islets. In the networks, islets were housed in a chamber that was continuously perfused with pressure-driven biological media at 0.6 microL min-1. Electroosmosis was used to pull perfusate containing secreted insulin into 4-cm-long reaction channels where it mixed with fluorescein isothiocyanate-labeled insulin and anti-insulin antibody for 60 s. The reaction streams were sampled at 6.25-s intervals and analyzed in parallel using an on-chip capillary electrophoresis separation with laser-induced fluorescence detection by a scanning confocal microscope. The limit of detection for insulin was 10 nM. The device was used to complete over 1450 immunoassays of biological samples in less than 40 min, allowing the parallel monitoring of insulin release from four islets every 6.25 s.     

An approach to multilayer microfluidic systems with integrated electrical, optical, and mechanical functionality

A general solution is presented for the realization of microfluidic systems containing multiple compartments with sequential functions. The approach utilizes multilayer plastic/glass/silicon microfluidic system configurations with integrated electrical, optical, and fluid control functionalities. The approach allows for the integration of on-column, on-chip electrical conductivity/impedance detectors and driving electrodes, as well as for the integration of on-column, off-chip optical detection using resonance light scattering. A process-compatible latex microvalve is demonstrated and characterized. The procedure utilizes a novel packaging technique for realizing integrated fluid and electrical interfaces between the microsystem and conventional tubing/instrumentation. The microfluidic system demonstrated in this work contains some of the most commonly used components in bioanalysis systems and can be used as a platform to fabricate more sophisticated microscale bioanalysis systems for a variety of applications.     

On-chip, time-correlated, fluorescence lifetime extraction algorithms and error analysis

A new, simple, and hardware-only fluorescence-lifetime-imaging microscopy (FLIM) is proposed to implement on-chip lifetime extractions, and their signal-to-noise-ratio based on statistics theory is also deduced. The results are compared with Monte Carlo simulations, giving good agreement. Compared with the commonly used iterative least-squares method or the maximum-likelihood-estimation- (MLE-) based, general purpose FLIM analysis software, our algorithm offers direct calculation of fluorescence lifetime based on the collected photon counts stored in on-chip counters and therefore delivers faster analysis for real-time applications, such as clinical diagnosis. Error analysis considering timing jitter based on statistics theory is carried out for the proposed algorithms and is also compared with MLE to obtain optimized channel width or measurement window and bit resolution of the time-to-digital converters for a given accuracy. A multi-exponential, pipelined fluorescence lifetime method based on the proposed algorithms is also introduced. The performance of the proposed methods has been tested on mono-exponential and four-exponential decay experimental data.     

A versatile multi-platform biochip surface attachment chemistry

A versatile DNA spotting and immobilization method for covalent attachment of amino-modified probe oligonucleotides in microarray format at glass, native silicon dioxide and CVD silicon nitride substrates is reported. Optimal probe spot printing and attachment buffers are identified for each substrate. Relative areal densities of immobilized probes as measured by epi-fluorescence microscopy vary with substrate type reflecting differences in surface morphology and chemistry. Target oligonucleotide hybridization occurs at glass and nitride supported probe microarrays in an efficient and reproducible manner with excellent measured fluorescence signal-to-background.     

fabrication of diamond membranes for MEMS using reactive ion etching of silicon

Polycrystalline diamond thin film has been grown on a silicon substrate using high pressure microwave plasma-assisted chemical vapor deposition from a gas mixture of methane and hydrogen at a substrate temperature of 950°C. A simple process flow has been developed to fabricate optically transparent polycrystalline synthetic diamond membranes/windows employing reactive ion etching (RIE) of a single crystal silicon substrate using an electron beam evaporated aluminum thin film mask pattern formed by photolithography. Scanning electron microscopy has been used to study the morphology of as-grown diamond thin films.     

Fabrication, characterization, and analysis of a DRIE CMOS-MEMS gyroscope

A gyroscope with a measured noise floor of 0.02/spl deg//s/Hz/sup 1/2/ at 5 Hz is fabricated by post-CMOS micromachining that uses interconnect metal layers to mask the structural etch steps. The 1 /spl times/ 1 mm lateral-axis angular rate sensor employs in-plane vibration and out-of-plane Coriolis acceleration detection with on-chip CMOS circuitry. The resultant device incorporates a combination of 1.8-/spl mu/m-thick thin-film structures for springs with out-of-plane compliance and 60-/spl mu/m-thick bulk silicon structures defined by deep reactive-ion etching for the proof mass and springs with out-of-plane stiffness. The microstructure is flat and avoids excessive curling, which exists in prior thin-film CMOS-microelectromechanical systems gyroscopes. Complete etch removal of selective silicon regions provides electrical isolation of bulk silicon to obtain individually controllable comb fingers. Direct motion coupling is observed and analyzed.     

Fabrication of diamond microstructures for microelectromechanical systems (MEMS) by a surface micromachining process

Selective polycrystalline diamond thin film has been grown on a silicon dioxide/silicon substrate using high pressure microwave plasma-assisted chemical vapor deposition from a gas mixture of methane and hydrogen at a substrate temperature of 950°C. A simple process flow has been developed to fabricate diamond microstructures such as diamond beams and cantilever beams using surface micromachining and photolithography for the first time. Scanning electron and optical microscopy has been used to characterize the surface micromachined diamond microstructures.     

Chip-based bioassay using bacterial sensor strains immobilized in three-dimensional microfluidic network

A whole-cell bioassay has been performed using Escherichia coli sensor strains immobilized in a chip assembly, in which a silicon substrate is placed between two poly(dimethylsiloxane) (PDMS) substrates. Microchannels fabricated on the two separate PDMS layers are connected via perforated microwells on the silicon chip, and thus, a three-dimensional microfluidic network is constructed in the assembly. Bioluminescent sensor strains mixed with agarose are injected into the channels on one of the two PDMS layers and are immobilized in the microwells by gelation. Induction of the firefly luciferase gene expression in the sensor strains can be easily carried out by filling the channels on the other layer with sample solutions containing mutagen. Bioluminescence emissions from each well are detected after injection of luciferin/ATP mixtures into the channels. In this assay format using two multichannel layers and one microwell array chip, the interactions between various types of samples and strains can be monitored at each well on one assembly in a combinatorial fashion. Using several genotypes of the sensor strains or concentrations of mitomycin C in this format, the dependence of bioluminescence on these factors was obtained simultaneously in the single screening procedure. The present method could be a promising on-chip format for high-throughput whole-cell bioassays.