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Huynh T.,University of Chicago | Sun B.,California Institute of Technology | Li L.,University of Chicago | Li L.,SlipChip | And 3 more authors.
Journal of the American Chemical Society | Year: 2013

In this article, we describe a nonlinear threshold chemistry based on enzymatic inhibition and demonstrate how it can be coupled with microfluidics to convert a chemical concentration (analog input) into patterns of ON or OFF reaction outcomes (chemical digital readout). Quantification of small changes in concentration is needed in a number of assays, such as that for cystatin C, where a 1.5-fold increase in concentration may indicate the presence of acute kidney injury or progression of chronic kidney disease. We developed an analog-to-digital chemical signal conversion that gives visual readout and applied it to an assay for cystatin C as a model target. The threshold chemistry is based on enzymatic inhibition and gives sharper responses with tighter inhibition. The chemistry described here uses acetylcholinesterase (AChE) and produces an unambiguous color change when the input is above a predetermined threshold concentration. An input gives a pattern of ON/OFF responses when subjected to a monotonic sequence of threshold concentrations, revealing the input concentration at the point of transition from OFF to ON outcomes. We demonstrated that this threshold chemistry can detect a 1.30-fold increase in concentration at 22 C and that it is robust to experimental fluctuations: it provided the same output despite changes in temperature (22-34 C) and readout time (10-fold range). We applied this threshold chemistry to diagnostics by coupling it with a traditional sandwich immunoassay for serum cystatin C. Because one quantitative measurement comprises several assays, each with its own threshold concentration, we used a microfluidic SlipChip device to process 12 assays in parallel, detecting a 1.5-fold increase (from 0.64 (49 nM) to 0.96 mg/L (74 nM)) of cystatin C in serum. We also demonstrated applicability to analysis of patient serum samples and the ability to image results using a cell phone camera. This work indicates that combining developments in nonlinear chemistries with microfluidics may lead to development of user-friendly diagnostic assays with simple readouts. © 2013 American Chemical Society.


Sun B.,California Institute of Technology | Shen F.,SlipChip | McCalla S.E.,California Institute of Technology | Kreutz J.E.,University of Washington | And 2 more authors.
Analytical Chemistry | Year: 2013

Here we used a SlipChip microfluidic device to evaluate the performance of digital reverse transcription-loop-mediated isothermal amplification (dRT-LAMP) for quantification of HIV viral RNA. Tests are needed for monitoring HIV viral load to control the emergence of drug resistance and to diagnose acute HIV infections. In resource-limited settings, in vitro measurement of HIV viral load in a simple format is especially needed, and single-molecule counting using a digital format could provide a potential solution. We showed here that when one-step dRT-LAMP is used for quantification of HIV RNA, the digital count is lower than expected and is limited by the yield of desired cDNA. We were able to overcome the limitations by developing a microfluidic protocol to manipulate many single molecules in parallel through a two-step digital process. In the first step we compartmentalize the individual RNA molecules (based on Poisson statistics) and perform reverse transcription on each RNA molecule independently to produce DNA. In the second step, we perform the LAMP amplification on all individual DNA molecules in parallel. Using this new protocol, we increased the absolute efficiency (the ratio between the concentration calculated from the actual count and the expected concentration) of dRT-LAMP 10-fold, from ∼2% to ∼23%, by (i) using a more efficient reverse transcriptase, (ii) introducing RNase H to break up the DNA:RNA hybrid, and (iii) adding only the BIP primer during the RT step. We also used this two-step method to quantify HIV RNA purified from four patient samples and found that in some cases, the quantification results were highly sensitive to the sequence of the patient's HIV RNA. We learned the following three lessons from this work: (i) digital amplification technologies, including dLAMP and dPCR, may give adequate dilution curves and yet have low efficiency, thereby providing quantification values that underestimate the true concentration. Careful validation is essential before a method is considered to provide absolute quantification; (ii) the sensitivity of dLAMP to the sequence of the target nucleic acid necessitates additional validation with patient samples carrying the full spectrum of mutations; (iii) for multistep digital amplification chemistries, such as a combination of reverse transcription with amplification, microfluidic devices may be used to decouple these steps from one another and to perform them under different, individually optimized conditions for improved efficiency. © 2013 American Chemical Society.


Begolo S.,California Institute of Technology | Shen F.,SlipChip | Ismagilov R.F.,California Institute of Technology
Lab on a Chip - Miniaturisation for Chemistry and Biology | Year: 2013

This paper describes a microfluidic device for dry preservation of biological specimens at room temperature that incorporates chemical stabilization matrices. Long-term stabilization of samples is crucial for remote medical analysis, biosurveillance, and archiving, but the current paradigm for transporting remotely obtained samples relies on the costly "cold chain" to preserve analytes within biospecimens. We propose an alternative approach that involves the use of microfluidics to preserve samples in the dry state with stabilization matrices, developed by others, that are based on self-preservation chemistries found in nature. We describe a SlipChip-based device that allows minimally trained users to preserve samples with the three simple steps of placing a sample at an inlet, closing a lid, and slipping one layer of the device. The device fills automatically, and a pre-loaded desiccant dries the samples. Later, specimens can be rehydrated and recovered for analysis in a laboratory. This device is portable, compact, and self-contained, so it can be transported and operated by untrained users even in limited-resource settings. Features such as dead-end and sequential filling, combined with a "pumping lid" mechanism, enable precise quantification of the original sample's volume while avoiding overfilling. In addition, we demonstrated that the device can be integrated with a plasma filtration module, and we validated device operations and capabilities by testing the stability of purified RNA solutions. These features and the modularity of this platform (which facilitates integration and simplifies operation) would be applicable to other microfluidic devices beyond this application. We envision that as the field of stabilization matrices develops, microfluidic devices will be useful for cost-effectively facilitating remote analysis and biosurveillance while also opening new opportunities for diagnostics, drug development, and other medical fields. © 2013 The Royal Society of Chemistry.


Patent
California Institute of Technology and SlipChip | Date: 2013-04-22

The present invention relates to fluidic devices for preparing, processing, storing, preserving, and/or analyzing samples. In particular, the devices and related systems and methods allow for preparing and/or analyzing samples (e.g., biospecimen samples) by using one or more of capture regions and/or automated analysis.


Patent
California Institute of Technology and SlipChip | Date: 2014-09-18

The present invention relates to fluidic systems for controlling one or more fluids and/or one or more reagents. These systems can be used in combination with one or more devices for assaying, processing, and/or storing samples. In particular, the systems and related methods can allow for dispensing fluid in a controlled manner and/or introducing pause(s) when implementing assays or processes.


Patent
SlipChip and California Institute of Technology | Date: 2013-04-24

The present invention relates to fluidic devices for compartmentalizing samples. In particular, the devices and related systems and methods allow for compartmentalization by using one or more first chambers connect by a first channel (e.g., where the cross-sectional dimension of the first channel is less than the cross-sectional dimension of at least one first chamber).


The present invention relates to fluidic systems for controlling one or more fluids or reagents. These systems can be used in combination with one or more devices for assaying, processing, or storing samples. In particular, the systems and related methods can allow for controlled pressure and actuation of fluids.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 170.15K | Year: 2011

DESCRIPTION (provided by applicant): Membrane proteins are crucial to a wide variety of physiological processes, functioning as chemical receptors, transport channels and signal transducers. It is estimated that over half of drug targets are membrane proteins. Structural knowledge of membrane proteins is of very high biomedical significance. Crystallization of membrane proteins is an important approach to obtaining structural information of membrane proteins. However, obtaining diffracting crystals of membrane proteins is still difficult due to a number of bottlenecks, including low quantity and poor stability of membrane proteins; search of larger parameter space; and difficulty to obtaining diffraction sufficient to determine structures. No technology is currently available to address all of the needs and bottlenecks. SlipChip is an attractive and innovative technology that can potentially achieve these goals. It has been already applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PI has demonstrated that SlipChip fabricated in glass can be used to handle and crystallize membrane proteins, and obtain crystals and high-resolution structures of soluble proteins. Yet, many technologies applicable to soluble proteins fail with membrane proteins; in addition, many elegant solutions to a single step in the crystallization workflow (e.g. establishing free interface diffusion) create additional bottlenecks elsewhere (e.g. creating problems in scale-up or crystal extraction). The goalof this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of membrane proteins, while testing the feasibility of upstream and downstream steps in the crystallization workflow: from Aim 1) simple loading thatwill meter out nL volumes robustly for a range of solutions without the need for any equipment, to Aim 2) performance, from filling to incubation and observation, of chips made in plastic by inexpensive molding techniques, to Aim 3) robust methods of extraction and diffraction of crystals. Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology for membrane protein crystallization, and would reduce the technical risk of Phase II work in this area, which would integrate many of the steps of concentration, separation, detergent exchange, purification, and formation of meso-phases into a single chip that accepts a fraction off a purification column and performs complex manipulations to produce diffraction-quality crystals of membrane proteins. PUBLIC HEALTH RELEVANCE: Structural knowledge of membrane proteins is of very high biomedical significance to public health. Crystallization of membrane proteins is an important approach to obtaining their structural information, but is difficult due to a number of bottlenecks. This proposal describes a SlipChip technology to address unmet needs and bottlenecks for membrane protein crystallization.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 173.38K | Year: 2011

DESCRIPTION (provided by applicant): 1. For the past 30 years, Sanger sequencing has enabled the determination of a sequence of nucleotide 2 bases on a small segment of DNA, with the crowning achievement of the technology being the complete 3 elucidationof the human genome at a cost of several hundreds of millions of dollars. Recently, next- 4 generation sequencing technologies have been developed capable of processing millions of DNA templates in 5 parallel. Compared to the astronomical cost of theoriginal human genome project, the current cost to 6 sequence a complete human genome using next-generation sequencing technology is currently under 7 50,000. However, even this relatively lower cost is still too high for the majority of scientific inquiry or clinical 8 studies that require deep sequencing of focused regions of the genome. Thus, targeted enrichment strategies 9 that enrich only desired segments of the genome for subsequent sequencing are necessary. And, there is 10 currently no solutionfor targeted enrichment exists that is simple, affordable to individual research or clinical 11 laboratories, and that incorporates quality control. 12 SlipChip is an attractive and innovative technology that can potentially solve this problem. SlipChip has 13 already been applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PIs 14 have demonstrated that SlipChips fabricated in glass can be used to perform multiplexed PCR and digital PCR. 15 The goal of this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of 16 multiplexed PCR for targeted enrichment and subsequent sequencing, while testing the feasibility of upstream 17 and downstream steps in the sequencing workflow: from Aim 1) where we will test and verify the robustness of 18 multiplexed PCR in the SlipChip using human DNA, Aim 2) where we will optimize the density of SlipChip and 19 finally, Aim 3) where We will verify that on-chip quality control of PCR before committing a sample is feasible. 20 Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology as a 21 sample processing tool for targeted enrichment for next generation sequencing by multiplexed PCR, and would 22 reduce the technical risk of Phase II work in this area, which would include sequencing experiments in the 23 context of biomedical problems of high significance, and will allow for the study of the validated SlipChip 24 system for complete integration in the workflow of an existing next-generation sequencing tool, specifically the 25 Illumina GA IIe. PUBLIC HEALTH RELEVANCE: 1 Technologies for the targeted enrichment of specific genomic regions are urgently needed to bring the 2 power of next-generation sequencing to fundamental and applied biomedical research. However, no solution 3 for targeted enrichment exists that is simple and affordable to individual research or clinical laboratories. This 4 proposal describes a SlipChip technology to address this unmet need.


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