Sumit Kalsi

Shaped apertures in photoresist films enhance the lifetime and mechanical stability of suspended lipid bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

Ultra-fast electronic detection of antimicrobial resistance genes using isothermal amplification and thin film transistor sensors

A low cost thin-film transistor (TFT) nanoribbon (NR) sensor has been developed for rapid real-time detection of DNA amplification using an isothermal Recombinase Polymerase Amplification (RPA) method. The semiconductor chip measures DNA amplification through a pH change, rather than via fluorescence. The utility of the method was demonstrated by amplifying CTX-M and NDM, two genes that confer bacterial resistance to cephalosporins and carbapenems, respectively. It is shown that this approach provides extremely fast and sensitive detection. It can detect <10 copies of the gene in genomic DNA extracted from E. coli or K. pneumoniae clinical isolates within a few minutes. A differential readout system was developed to minimize the effect of primer-dimer amplification on the assay. The simple device has the potential for low cost, portable and real-time nucleic acid analysis as a Point of Care device.

A Programmable Digital Microfluidic Assay for the Simultaneous Detection of Multiple Anti-Microbial Resistance Genes

The rapid emergence of antimicrobial resistant bacteria requires the development of new diagnostic tests. Nucleic acid-based assays determine antimicrobial susceptibility by detecting genes that encode for the resistance. In this study, we demonstrate rapid and simultaneous detection of three genes that confer resistance in bacteria to extended spectrum β-lactam and carbapenem antibiotics; CTX-M-15, KPC and NDM-1. The assay uses isothermal DNA amplification (recombinase polymerase amplification, RPA) implemented on a programmable digital microfluidics (DMF) platform. Automated dispensing protocols are used to simultaneously manipulate 45 droplets of nL volume containing sample DNA, reagents, and controls. The droplets are processed and mixed under electronic control on the DMF devices with positive amplification measured by fluorescence. The assay on these devices is significantly improved with a Time to Positivity (TTP) half that of the benchtop assay.

Integrated waveguide and nanostructured sensor platform for surface-enhanced Raman spectroscopy

Abstract. Limitations of current sensors include large dimensions, sometimes limited sensitivity and inherent single-parameter measurement capability. Surface-enhanced Raman spectroscopy can be utilized for environment and pharmaceutical applications with the intensity of the Raman scattering enhanced by a factor of 106. By fabricating and characterizing an integrated optical waveguide beneath a nanostructured precious metal coated surface a new surface-enhanced Raman spectroscopy sensing arrangement can be achieved. Nanostructured sensors can provide both multiparameter and high-resolution sensing. Using the slab waveguide core to interrogate the nanostructures at the base allows for the emission to reach discrete sensing areas effectively and should provide ideal parameters for maximum Raman interactions. Thin slab waveguide films of silicon oxynitride were etched and gold coated to create localized nanostructured sensing areas of various pitch, diameter, and shape. These were interrogated using a Ti:Sapphire laser tuned to 785-nm end coupled into the slab waveguide. The nanostructured sensors vertically projected a Raman signal, which was used to actively detect a thin layer of benzyl mercaptan attached to the sensors.

Fast and sensitive isothermal DNA assay using microbead dielectrophoresis for detection of anti-microbial resistance genes

Antimicrobial resistant pathogens are a growing worldwide threat to human health. This study describes a novel method for rapid and sensitive detection of antimicrobial resistance (AMR) genes, specifically blaCTX-M-15 which encodes for the enzyme that offers resistance to extended spectrum β-lactam antibiotics. The method combines isothermal DNA amplification by recombinase polymerase amplification (RPA), with microbead dielectrophoresis (DEP)-based DNA detection. The RPA amplicon is captured onto dielectric microbeads, and the amount of amplicon determined by dielectrophoretic impedance measurement (DEPIM) of the microbeads. Amplicon-labeled microbeads were prepared by either a two-step or one-step method. A purified recombinant plasmid containing blaCTX-M-15 and genomic DNA (with plasmid) extracted from an AMR bacteria (Escherichia coli NCTC 13441) were used as target samples. A one-step method in which RPA and DNA immobilization on the microbeads is carried out simultaneously, has a detection limit of 2 copies/reaction for pure plasmid and 50 copies/reaction for genomic DNA. The assays are quantitative with a dynamic range up to 105 copies/reaction, with a total detection time of 26 min. Both methods are easy, rapid, and unlike lateral flow detection are quantitative.

Waveguide core integrated nanostructured SERS sensor platform

The intensity of Raman scattering can be enhanced by a factor of 106 using Surface Enhanced Raman Spectroscopy (SERS). In this method, molecules are placed within a few nm of a rough/nanostructured metal surface. In this paper we show fabrication and characterisation of an integrated optical waveguide beneath a nano-structured precious metal coated surface. By using a waveguide core, the excitation field comes from underneath and enters the nanostructures at the base. This allows the emission to reach the discrete sensing areas effectively and should provide ideal parameters for maximum Raman interactions. The nanostructured geometry projects the Plasmon field into free space, thus increasing the cross section of interaction between the analyte molecules and optical fields, thereby increasing device sensitivity. Thin films of silicon oxynitride were deposited using PECVD on to thermal oxide coated 4 inch wafers and annealed at various temperatures to obtain low loss layers suitable for the waveguide core material. Based on the results from our simulations, nanostructured features of various diameters/feature lengths and pitch were etched into the low loss silicon oxynitride layer. The sensor area was coated with a thin layer of gold (25nm) and a variety of optical measurements were completed for many of the processed test chips including broadband reflectrometry, normal incident Raman spectroscopy and waveguide Raman spectroscopy using a Raman probe above the sensor area. The results showed that detection of a Raman active molecule (Benzyl Mercaptan) was possible when excited from the underlying waveguide core with 104 sensitivity.