Anomalous diffusion of E. coli under microfluidic confinement and chemical gradient.

We report a two-layer microfluidic device to study the combined effect of confinement and chemical gradient on the motility of wild-type E. coli. We track individual E. coli in 50 µm and 10 µm wide microchannels, with a channel height of 2 µm, to generate quasi-2D conditions. We find that contrary to expectations, bacterial trajectories are superdiffusive even in the absence of a chemical (glucose) gradient. The superdiffusive behaviour becomes more pronounced upon introducing a chemical gradient or strengthening the lateral confinement. Run length distributions for weak lateral confinement in the absence of chemical gradients follow an exponential distribution. Both confinement and chemoattraction induce deviations from this behaviour, with the run length distributions approaching a power-law form under these conditions. Both confinement and chemoattraction suppress large-angle tumbles as well. Our results suggest that wild-type E. coli modulates both its runs and tumbles in a similar manner under physical confinement and chemical gradient. Our findings have implications for understanding how bacteria modulate their motility behaviour in natural habitats.

Effect of microchannel geometry and linker molecules on surface immobilization efficiency of proteins in microfluidic devices.

One of the key metrics in the design of biosensors is the presence of an effective capture layer. Surface-immobilized proteins (especially as a part of antibody-antigen combinations) are the most commonly used capture ligands in biosensors. The surface coverage of these proteins in flow-based biosensors are affected by both the linker chemistry used to attach them as well as the microchannel geometry. We used streptavidin as a model protein to compare glutaraldehyde, EDC-NHS, sulfo-SMCC and sulfo-NHS-biotin as linkers inside straight, serpentine and square-wave microchannel geometries. We found that straight microchannels achieve the highest degree of protein immobilization compared to serpentine and square-wave microchannels, irrespective of the linker chemistry used. We also showed that for a given microchannel geometry, sulfo-NHS-biotin leads to the highest immobilization of streptavidin among all the linkers.

Development of a PNA-DiSc2 based portable absorbance platform for the detection of pathogen nucleic acids.

We report the development of a portable absorption (PortAbs)-based pathogen nucleic acid detection system using peptide nucleic acid (PNA) and a cyanine dye, DiSc2(5). When the dye binds to the PNA-DNA hybrid, it results in a characteristic ∼110 nm shift in the dye absorbance, which we measure using PortAbs. The protocol involves amplification of the target DNA, PNA-DNA hybridization and dye complexing steps followed by absorption measurement. The system is built using a broad-spectrum photodiode whose output is amplified and then measured by a high resolution (24 or 32 bit) analog-to-digital converter. The excitation pulses of light are delivered by a color-changing LED. The sequence of excitation, measurement and display of results are all controlled by an embedded Raspberry-Pi board (or alternatively a laptop). At higher concentrations of the target amplicon (∼200 ng), the color change can be detected visually. At lower concentrations, PortAbs outperforms a plate reader and can detect target DNA as low as 30 ng or approximately 10 nM which is at least 10 fold better than previously reported studies. We validate the methodology using SARS-CoV-2 clinical samples containing about 1000 copies of the viral RNA and show that the entire workflow takes about 90 min. The cost of the complete standalone system is less than INR 40 000 (approx. 500 USD).

Development of an insilico model of eccrine sweat using molecular modelling techniques.

Eccrine sweat is an ideal surrogate diagnostic biofluid for physiological and metabolic biomarkers for wearable biosensor design. Its periodic and non-invasive availability for candidate analytes such as glucose and cortisol along with limited correlation with blood plasma is of significant research interest. An insilico model of eccrine sweat can assist in the development of such wearable biosensors. In this regard, molecular modelling can be employed to observe the most fundamental interactions. Here, we determine a suitable molecular model for building eccrine sweat. The basic components of sweat are water and sodium chloride, in which glucose and other analytes are present in trace quantities. Given the wide range of water models available in the molecular dynamics space, in this study, we first validate the water models. We use three compounds to represent the base to build bulk sweat fluid and validate the force fields. We compare the self-diffusivity of water, glucose, sodium, and chloride ions as well as bulk viscosity values and present the results which are > 90% accurate as compared with the available literature. This validated insilico eccrine sweat model can serve as an aid to expedite the development de novo biosensors by addition of other analytes of interest e.g. cortisol, uric acid etc., simulate various temperatures and salt concentrations, expand search space for screening candidate target receptors by their binding affinity and assess the interference between competing species via simulations.

Eccrine Sweat Molecular Model for Development of de novo Biosensors.

In this paper, we present a validated, novel, in silico molecular dynamics (MD) model of eccrine sweat with approx. 35k atoms developed using Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) program. CHARMMS36m force field for constituent atoms and SPC/E water model are used to develop this model. The model outputs transport properties such as self-diffusivity computed using mean squared displacement and bulk viscosity computed via Green-Kubo correlations, which are compared with existing literature values and experimental studies and presented. This validated model is intended to serve as a tool to develop eccrine sweat based biosensors.

Temperature-dependent self-assembly of biofilaments during red blood cell sickling.

Molecular self-assembly plays a vital role in various biological functions. However, when aberrant molecules self-assemble to form large aggregates, it can give rise to various diseases. For example, sickle cell disease and Alzheimer's disease are caused by self-assembled hemoglobin fibers and amyloid plaques, respectively. Here, we study the assembly kinetics of such fibers using kinetic Monte Carlo simulation. We focus on the initial lag time of these highly stochastic processes, during which self-assembly is very slow. The lag time distributions turn out to be similar for two very different regimes of polymerization, namely, (a) when polymerization is slow and depolymerization is fast and (b) the opposite case, when polymerization is fast and depolymerization is slow. Using temperature-dependent on- and off-rates for hemoglobin fiber growth, reported in recent in vitro experiments, we show that the mean lag time can exhibit non-monotonic behavior with respect to the change in temperature.

Haemoprocessor: A Portable Platform Using Rapid Acoustically Driven Plasma Separation Validated by Infrared Spectroscopy for Point-of-Care Diagnostics.

The identification of biomarkers from blood plasma is at the heart of many diagnostic tests. These tests often need to be conducted frequently and quickly, but the logistics of sample collection and processing not only delays the test result, but also puts a strain on the healthcare system due to the sheer volume of tests that need to be performed. The advent of microfluidics has made the processing of samples quick and reliable, with little or no skill required on the user's part. However, while several microfluidic devices have been demonstrated for plasma separation, none of them have validated the chemical integrity of the sample post-process. Here, we present Haemoprocessor: a portable, robust, open-fluidic system that utilizes Travelling Surface Acoustic Waves (TSAW) with the expression of overtones to separate plasma from 20× diluted human blood within a span of 2 min to achieve 98% RBC removal. The plasma and red blood cell separation quality/integrity was validated through Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy and multivariate analyses to ascertain device performance and reproducibility when compared to centrifugation (the prevailing gold-standard for plasma separation). Principal Component Analysis (PCA) showed a remarkable separation of 92.21% between RBCs and plasma components obtained through both centrifugation and Haemoprocessor methods. Moreover, a close association between plasma isolates acquired by both approaches in PCA validated the potential of the proposed system as an eminent cell enrichment and plasma separation platform. Thus, compared to contemporary acoustic devices, this system combines the ease of operation, low sample requirement of an open system, the versatility of a SAW device using harmonics, and portability.

Dynamic generation of power function gradient profiles in a universal microfluidic gradient generator by controlling the inlet flow rates.

We report a two-inlet universal microfluidic gradient generator capable of generating gradient profiles of the functional form xp in the same device by controlling only the inlet flow rates. We have developed an analytical model to predict the inlet flow rates needed to generate a user-specified gradient profile at the outlet. We have validated this model by performing both COMSOL simulations and experiments. Our experiments show an excellent match between the target functions (x0.33, x1, x2 and x3) and the gradient profiles generated in this device. Unlike the universal gradient generators reported earlier, our device does not require changing the positions of the internal barriers for each new gradient profile, thereby making it easier for the user to operate this device.

Developing a Point-of-Care Molecular Test to Detect SARS-CoV-2.

There is a need for widespread testing in India to stop the spread of the novel coronavirus in the population. While RT-PCR is the recommended diagnostic technique, its use is limited to well-equipped laboratories due to the need for specialized instrumentation, reagents and trained personnel. Immunodiagnostic tests are not yet recommended by the WHO for diagnosing active infections. There is a strong need for developing point-of-care molecular tests. Based on our past experience with paperfluidic devices for diagnosing bacterial infections by molecular tests, we propose the development of a diagnostic test for COVID-19. As a platform technology, it could be adapted to other viral outbreaks in future.

Label-Free Electrochemical Detection of S. mutans Exploiting Commercially Fabricated Printed Circuit Board Sensing Electrodes.

This paper reports for the first time printed-circuit-board (PCB)-based label-free electrochemical detection of bacteria. The demonstrated immunosensor was implemented on a PCB sensing platform which was designed and fabricated in a standard PCB manufacturing facility. Bacteria were directly captured on the PCB sensing surface using a specific, pre-immobilized antibody. Electrochemical impedance spectra (EIS) were recorded and used to extract the charge transfer resistance (Rct) value for the different bacteria concentrations under investigation. As a proof-of-concept, Streptococcus mutans (S. mutans) bacteria were quantified in a phosphate buffered saline (PBS) buffer, achieving a limit of detection of 103 CFU/mL. Therefore, the proposed biosensor is an attractive candidate for the development of a simple and robust point-of-care diagnostic platform for bacteria identification, exhibiting good sensitivity, high selectivity, and excellent reproducibility.

Clogging-free continuous operation with whole blood in a radial pillar device (RAPID).

Pillar-based passive microfluidic devices combine the advantages of simple designs, small device footprint, and high selectivity for size-based separation of blood cells. Most of these device designs have been validated with dilute blood samples. Handling whole blood in pillar-based devices is extremely challenging due to clogging. The high proportion of cells (particularly red blood cells) in blood, the varying sizes and stiffness of the different blood cells, and the tendency of the cells to aggregate lead to clogging of the pillars within a short period. We recently reported a ra dial pi llar d evice (RAPID) design for continuous and high throughput separation of multi-sized rigid polystyrene particles in a single experiment. In the current manuscript, we have given detailed guidelines to modify the design of RAPID for any application with deformable objects (e.g. cells). We have adapted RAPID to work with whole blood without any pre-processing steps. We were successful in operating the device with whole blood for almost 6 h, which is difficult to achieve with most pillar-based devices. The availability of multiple parallel paths for the cells and the provision for a self-generating cross flow in the device design were the main reasons behind the minimal clogging in our device. We also observed that a vibrator motor attached to the inlet tubing occasionally disturbed the cell clumps. As an illustration of the improved device design, we demonstrated up to ∼ 60-fold enrichment of platelets.

Fabrication of miniature elastomer lenses with programmable liquid mold for smartphone microscopy: curing polydimethylsiloxane with in situ curvature control.

Miniature lenses can transform commercial imaging systems, e.g., smartphones and webcams, into powerful, low-cost, handheld microscopes. To date, the reproducible fabrication of polymer lenses is still a challenge as they require controlled dispensing of viscous liquid. This paper reports a reproducible lens fabrication technique using liquid mold with programmable curvature and off-the-shelf materials. The lens curvature is controlled during fabrication by tuning the curvature of an interface of two immiscible liquids [polydimethylsiloxane (PDMS) and glycerol]. The curvature control is implemented using a visual feedback system, which includes a software-based guiding system to produce lenses of desired curvature. The technique allows PDMS lens fabrication of a wide range of sizes and focal lengths, within 20 min. The fabrication of two lens diameters: 1 and 5 mm with focal lengths ranging between 1.2 and 11 mm are demonstrated. The lens surface and bulk quality check performed using X-ray microtomography and atomic force microscopy reveal that the lenses are suitable for optical imaging. Furthermore, a smartphone microscope with ∼1.4-µm resolution is developed using a self-assembly of a single high power fabricated lens and microaperture. The lenses have various potential applications, e.g., optofluidics, diagnostics, forensics, and surveillance.

A Radial Pillar Device (RAPID) for continuous and high-throughput separation of multi-sized particles.

Pillar-based microfluidic sorting devices are preferred for isolation of rare cells due to their simple designs and passive operation. Dead-end pillar filters can efficiently capture large rare cells, such as, circulating tumor cells (CTCs), nucleated red blood cells (NRBCs), CD4 cells in HIV patients, etc., but they get clogged easily. Cross flow filters are preferred for smaller rare particles (e.g. separating bacteria from blood), but they need additional buffer inlets and a large device footprint for efficient operation. We have designed a new microparticle separation device i.e. Ra dial Pi llar D evice (RAPID) that combines the advantages of dead-end and cross flow filters. RAPID can simultaneously isolate both large and small rare particles from a mixed population, while functioning for several hours without clogging. We have achieved simultaneous separation of 10 µ m and 2 µ m polystyrene particles from a mixture of 2 µ m, 7 µ m and 10 µ m particles. RAPID achieved average separation purity and recovery in excess of ∼90%. The throughput of our device (∼3ml/min) is 10 and 100 times higher compared to cross flow and dead-end filters respectively, thereby justifying the name RAPID.

Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube.

Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65°C and 60min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.

Phagocytosis dynamics depends on target shape.

A complete understanding of phagocytosis requires insight into both its biochemical and physical aspects. One of the ways to explore the physical mechanism of phagocytosis is to probe whether and how the target properties (e.g., size, shape, surface states, stiffness, etc.) affect their uptake. Here we report an imaging-based method to explore phagocytosis kinetics, which is compatible with real-time imaging and can be used to validate existing reports using fixed and stained cells. We measure single-event engulfment time from a large number of phagocytosis events to compare how size and shape of targets determine their engulfment. The data shows an increase in the average engulfment time for increased target size, for spherical particles. The uptake time data on nonspherical particles confirms that target shape plays a more dominant role than target size for phagocytosis: Ellipsoids with an eccentricity of 0.954 and much smaller surface areas than spheres were taken up five times more slowly than spherical targets.

Transferrin-mediated rapid targeting, isolation, and detection of circulating tumor cells by multifunctional magneto-dendritic nanosystem.

A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer.

Enhancing surface interactions with colon cancer cells on a transferrin-conjugated 3D nanostructured substrate.

A transferrin-conjugated PEG-Fe(3) O(4) nanostructured matrix is developed to explore cellular responses in terms of enhanced cell adhesion, specific interactions between ligands in the matrix and molecular receptors on the cell membrane, comparison of cell shapes on 2D and 3D surfaces, and effect of polymer architecture on cell adhesion. Integration of such advanced synthetic nanomaterials into a functionalized 3D matrix to control cell behavior on surfaces will have implications in nanomedicine.

A "dry and wet hybrid" lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing.

A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a "dry and wet hybrid" technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl). We show that the two lithography protocols are chemically compatible with each other. Finally, we demonstrate the hybrid technique in two different microfluidic applications: (1) a neuron culture device with compartmentalization of different elements of a neuron and (2) a two-phase (gas-liquid) global micromixer for fast mixing of a small amount of a viscous liquid into a larger volume of a less viscous liquid.

Label-free sub-picomolar protein detection with field-effect transistors.

Proteins mediate the bulk of biological activity and are powerfully assayed in the diagnosis of diseases. Protein detection relies largely on antibodies, which have significant technical limitations especially when immobilized on two-dimensional surfaces. Here, we report the integration of peptide aptamers with extended gate metal-oxide-semiconductor field-effect transistors (MOSFETs) to achieve label-free sub-picomolar target protein detection. Specifically, peptide aptamers that recognize highly related protein partners of the cyclin-dependent kinase (CDK) family are immobilized on the transistor gate to enable human CDK2 to be detected at 100 fM or 5 pg/mL, well within the clinically relevant range. The target specificity, ease of fabrication, and scalability of these FET arrays further demonstrate the potential application of the multiplexable field effect format to protein sensing.

Surface-immobilized peptide aptamers as probe molecules for protein detection.

We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.