Multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip.

The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.

An array-based melt curve analysis method for the identification and classification of closely related pathogen strains.

PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5'-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.

A Fully Integrated CMOS Fluorescence Biochip for DNA and RNA Testing.

Design and successful implementation of a fully-integrated CMOS fluorescence biochip for DNA/RNA testing in molecular diagnostics (MDx) is presented. The biochip includes a 32×32 array of continuous wave fluorescence detection biosensing elements. Each biosensing element is capable of having unique DNA probe sequences, wavelength-selective multi-dielectric emission filter (OD of 3.6), resistive heater for thermal cycling, and a high performance and programmable photodetector. The dimension of each biosensor is 100µm×100µm with a 50µm×50µm Nwell-Psub photodiode acting as the optical transducer, and a ΣΔ modulator based photocurrent sensor. The measured photodetector performance shows ~116 dB detection dynamic range (10fA - 10nA) over the 25°C - 100°C temperature range, while being ~1 dB away from the fundamental shot-noise limit. To empirically demonstrate the compatibility of this biochip with MDx applications, we have successfully utilized the array and its thermal cycling capability to adopt a 7-plex panel for detection of 6 human upper respiratory viruses.

Interface design for CMOS-integrated Electrochemical Impedance Spectroscopy (EIS) biosensors.

Electrochemical Impedance Spectroscopy (EIS) is a powerful electrochemical technique to detect biomolecules. EIS has the potential of carrying out label-free and real-time detection, and in addition, can be easily implemented using electronic integrated circuits (ICs) that are built through standard semiconductor fabrication processes. This paper focuses on the various design and optimization aspects of EIS ICs, particularly the bio-to-semiconductor interface design. We discuss, in detail, considerations such as the choice of the electrode surface in view of IC manufacturing, surface linkers, and development of optimal bio-molecular detection protocols. We also report experimental results, using both macro- and micro-electrodes to demonstrate the design trade-offs and ultimately validate our optimization procedures.

A CMOS Electrochemical Impedance Spectroscopy (EIS) Biosensor Array.

In this paper, we present a fully integrated biosensor 10 × 10 array in a standard complementary metal-oxide semiconducor process, which takes advantage of electrochemical impedance spectroscopy (EIS). We also show that this system is able to detect various biological analytes, such as DNA and proteins, in real time and without the need for molecular labels. In each pixel of this array, we implement a biocompatible Au electrode transducer and embedded sensor circuitry which takes advantage of the coherent detector to measure the impedance of the associated electrode-electrolyte interface. This chip is capable of concurrently measuring admittance values as small as 10(-8) Ω(-1) within the array with the detection dynamic range of more than 90 dB in the frequency range of 10 Hz-50 MHz.