Govind V. Kaigala

Rapid prototyping of microfluidic devices with a wax printer

We demonstrate a rapid and inexpensive approach for the fabrication of high resolution poly(dimethylsiloxane) (PDMS)-based microfluidic devices. The complete process of fabrication could be performed in several hours (or less) without any specialized equipment other than a consumer-grade wax printer. The channels produced by this method are of high enough quality that we are able to demonstrate the sizing and separation of DNA fragments using capillary electrophoresis (CE) with no apparent loss of resolution over that found with glass chips fabricated by conventional photolithographic methods. We believe that this method will greatly improve the accessibility of rapid prototyping methods.

Rapid detection of urinary tract infections using isotachophoresis and molecular beacons

We present a novel assay for rapid detection and identification of bacterial urinary tract infections using isotachophoresis (ITP) and molecular beacons. We applied on-chip ITP to extract and focus 16S rRNA directly from bacterial lysate and used molecular beacons to achieve detection of bacteria specific sequences. We demonstrated detection of E. coli in bacteria cultures as well as in patient urine samples in the clinically relevant range 1E6-1E8 cfu/mL. For bacterial cultures we further demonstrate quantification in this range. The assay requires minimal sample preparation (a single centrifugation and dilution), and can be completed, from beginning of lysing to detection, in under 15 min. We believe that the principles presented here can be used for design of other rapid diagnostics or detection methods for pathogenic diseases.

High‐sensitivity detection using isotachophoresis with variable cross‐section geometry

We present a theoretical and experimental study on increasing the sensitivity of ITP assays by varying channel cross‐section. We present a simple, unsteady, diffusion‐free model for plateau mode ITP in channels with axially varying cross‐section. Our model takes into account detailed chemical equilibrium calculations and handles arbitrary variations in channel cross‐section. We have validated our model with numerical simulations of a more comprehensive model of ITP. We show that using strongly convergent channels can lead to a large increase in sensitivity and simultaneous reduction in assay time, compared to uniform cross‐section channels. We have validated our theoretical predictions with detailed experiments by varying channel geometry and analyte concentrations. We show the effectiveness of using strongly convergent channels by demonstrating indirect fluorescence detection with a sensitivity of 100 nM. We also present simple analytical relations for dependence of zone length and assay time on geometric parameters of strongly convergent channels. Our theoretical analysis and experimental validations provide useful guidelines on optimizing chip geometry for maximum sensitivity under constraints of required assay time, chip area and power supply.

Elemental analysis using micro Laser-induced Breakdown Spectroscopy (µLIBS) in a microfluidic platform

We present here a non-labeled, elemental analysis detection technique that is suitable for microfluidic chips, and demonstrate its applicability with the sensitive detection of sodium (Na). Spectroscopy performed on small volumes of liquids can be used to provide a true representation of the composition of the isolated fluid. Performing this using low power instrumentation integrated with a microfluidic platform makes it potentially feasible to develop a portable system. For this we present a simple approach to isolating minute amounts of fluid from bulk fluid within a microfluidic chip. The chip itself contains a patterned thin film resistive element that super-heats the sample in tens of microseconds, creating a micro-bubble that extrudes a micro-droplet from the microchip. For simplicity a non-valved microchip is used here as it is highly compatible to a continuous flow-based fluidic system suitable for continuous sampling of the fluid composition. We believe such a nonlabeled detection technique within a microfluidic system has wide applicability in elemental analysis. This is the first demonstration of laser-induced breakdown spectroscopy (LIBS) as a detection technology in conjunction with microfluidics, and represents first steps towards realizing a portable lower power LIBS-based detection system.

Inexpensive, universal serial bus-powered and fully portable lab-on-a-chip-based capillary electrophoresis instrument

Capillary electrophoresis is a cornerstone of lab-on-a-chip (LOC) implementations for medical diagnostics. However, the infrastructure needed to operate electrophoretic LOC implementations tends to be large and expensive, hindering the development of portable or low-cost systems. A custom-designed and highly integrated microelectronic chip for high-voltage generation switching and interfacing is recently developed. Here, the authors integrate the microelectronic chip with a microfluidic chip, a solid-state laser, filter, lens and several dollars worth of electronic components to form an inexpensive and portable platform, which is the size of a mobile telephone. This compact system has such reduced power requirements that the complete platform can be operated using a universal serial bus link to a computer. It is believed that this system represents a significant advancement in practical LOC implementations for point-of-care medical diagnostics.

Fluorescent Carrier Ampholytes Assay for Portable, Label-Free Detection of Chemical Toxins in Tap Water

We present a novel method for fluorescence-based indirect detection of analytes and demonstrate its use for label-free detection of chemical toxins in a hand-held device. We fluorescently label a mixture of low-concentration carrier ampholytes and introduce it into an isotachophoresis (ITP) separation. The carrier ampholytes provide a large number of fluorescent species with a wide range of closely spaced effective electrophoretic mobilities. Analytes focus under ITP and displace subsets of these carrier ampholytes. The analytes are detected indirectly and quantified by analyzing the gaps in the fluorescent ampholyte signal. The large number (on the order of 1000) of carrier ampholytes enables detection of a wide range of analytes, requiring little a priori knowledge of their electrophoretic properties. We discuss the principles of the technique and demonstrate its use in the detection of various analytes using a standard microscope system. We then present the integration of the technique into a self-contained hand-held device and demonstrate detection of chemical toxins (2-nitrophenol and 2,4,6-trichlorophenol) in tap water, with no sample preparation steps.

Method for Analyte Identification Using Isotachophoresis and a Fluorescent Carrier Ampholyte Assay

We present a novel method for identification of unlabeled analytes using fluorescent carrier ampholytes and isotachophoresis (ITP). The method is based on previous work where we showed that the ITP displacement of carrier ampholytes can be used for detection of unlabeled (nonfluorescent) analytes. We here propose a signal analysis method based on integration of the associated fluorescent signal. We define a normalized signal integral which is equivalent to an accurate measure of the amount of carrier ampholytes which are focused between the leading electrolyte and the analyte. We show that this parameter can be related directly to analyte effective mobility. Using several well characterized analytes, we construct calibration curves relating effective mobility and carrier ampholyte displacement at two different leading electrolyte (LE) buffers. On the basis of these calibration curves, we demonstrate the extraction of fully ionized mobility and dissociation constant of 2-nitrophenol and 2,4,6-trichlorophenol from ITP experiments with fluorescent carrier ampholytes. This extraction is based on no a priori assumptions or knowledge of these two toxic chemicals. This technique allows simultaneous identification of multiple analytes by their physiochemical properties in a few minutes and with no sample preparation.

Nonlinear Controller Designs for Thermal Management in PCR Amplification

We have developed a theoretical model for a cascade two-stage Peltier device based on the electrothermal dynamics of Peltier modules and the heat balance equations of the interfacing materials. Both open- and closed-loop data are used to tune the scaling factors of the nonlinear model. The effectiveness of the model is validated over a large temperature range with the experimental data from a thermal cycling application of the Peltier device used to perform the polymerase chain reaction (PCR), a genetic amplification technique having important medical diagnostic applications. Based on the theoretical model, two novel nonlinear controllers are designed for a PCR cycling temperature profile. The first controller is an extension of conventional input-to-state feedback linearization design to a class of nonlinear systems that is not only affine on the control but also affine on the square of control inputs. The desired performance is achieved by tuning the parameters to control the convergent rates of the tracking errors. The second one is a switching controller design, which switches between a nonlinear pseudo-proportional-integral-differential (PID)/state feedback controller and a linear time-invariant proportional-integral (PI)/state feedback controller. A Lyapunov function method is used to develop the algorithm for the nonlinear controller, whose parameter values at the switching time are used in the linear controller. Such a combination of linear and nonlinear controllers could reduce the calculation burden and minimize the steady-state errors. Both controllers are tested with our simulation model and implemented in a microcontroller. We verified the designs with improved temperature tracking performances compared to our earlier linear switching design on reduced overshoots (<; 0.5 °C) and settling time (8-10 s faster). The modeling methodology and the feedback linearization-based controller design are scalable and both nonlinear designs can avoid future local model identifications when applied to different references, therefore, are easily extended to other thermal applications.

Readily integrated, electrically controlled microvalves

We present a simple method for fabricating and operating normally open, electrothermally actuated microvalves. These valves are fabricated by placing a gas-permeable elastomeric membrane between two etched glass plates. The reservoirs and channels on one layer are filled with a low melting point polymer (polyethylene glycol, PEG) that exhibits a large volumetric change (of up to 30%) upon phase transition (melting). This volume expansion is used to actuate the membrane and seal the microfluidic channels located in the second etched glass plate. The PEG in the reservoir is heated with integrated patterned platinum-resistive elements. The valve reliably seals the microfluidic channel against external fluid pressures of 10 psi. This valve can be readily integrated with one of the standard technologies for lab-on-a-chip (LOC) fabrication and is suitable for use with the polymerase chain reaction. The novelty of this microvalve lies in the ability to fill dead-end microchannels with a polymer, its self-sealing ability, the ability to remotely actuate the valve by transferring pressure via a microchannel and the compatibility of this microvalve with standard LOC technologies.

Strategies for enhancing the speed and integration of microchip genetic amplification

In this work, we explore the use of methods that allow a significant acceleration of genetic analysis within microchips fabricated from low thermal conductivity materials such as glass or polymers. Although these materials are highly suitable for integrating a number of genetic analysis techniques onto lab‐on‐a‐chip devices, their low thermal conductivity limits the rate at which heat can be transferred and hence lowers the speed of thermal cycling. However, short thermal cycling times are the key to bringing PCR to clinical point‐of‐care applications. Although shrinking the PCR reaction chamber volume can increase the speed of thermal cycling, this strategy is not always suitable, particularly when dealing with clinical samples with low analyte concentrations. In the present work, we combine two alternate strategies for decreasing the time required to perform PCR: implementing a heat sink and optimizing the PCR protocol. First, the heat sink substantially reduces the thermal resistance opposing heat dissipation into the ambient environment, and eliminates the parasitic thermal capacitance of the regions in the microchip that do not require heating. The low thermal conductivity of glass is used to our advantage to design the heat‐sink placement to achieve fast thermal transitions while maintaining low power consumption. Second, we explore the application of two‐stage PCR to provide a further reduction in the time required to perform genetic amplification by merging the annealing and extension stages of the commonly used three‐stage PCR approach. In combination, we reduce the time required to perform thermal cycling by roughly a factor of 3 while improving the temperature control.