The discovery of the RNA-guided DNA nuclease CRISPR-Cas9 has enabled the targeted editing of genomes from diverse organisms, but the permanent and inheritable nature of genome modification also poses immense risks. The potential for accidental exposure, malicious use, or undesirable persistence of Cas9 therapeutics and off-target genome effects highlight the need for detection assays. Here we report a centrifugal microfluidic platform for the measurement of both Cas9 protein levels and nuclease activity. Because Cas9 from many bacterial species have been adapted for biotechnology applications, we developed the capability to detect Cas9 from the widely-used S. pyogenes, as well as S. aureus, N. meningitidis, and S. thermophilus using commercially-available antibodies. Further, we show that the phage-derived anti-CRISPR protein AcrIIC1, which binds to Cas9 from several species, can be used as a capture reagent to broaden the species range of detection. As genome modification generally requires Cas9 nuclease activity, a fluorescence-based sedimentation nuclease assay was also incorporated to allow the sensitive and simultaneous measurement of both Cas9 protein and activity in a single biological sample.
The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection = 1.35–5.50 ng/mL for immunoassays and 1–30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.
Infection with Mycobacterium Tuberculosis represents a significant threat to people with immune disorders, such as HIV-positive individuals, and can result in significant health complications or death if not diagnosed and treated early. We present a centrifugal microfluidic platform for multiplexed detection of tuberculosis and HIV biomarkers in human whole blood with minimal sample preparation and a sample-to-answer time of 30 minutes. This multiplexed assay was developed for the detection of two M.tuberculosis secreted proteins, whose secretion represents an active and ongoing infection, as well as detection of HIV p24 protein and human anti-p24 antibodies. The limit of detection for this multiplex assay is in the pg/mL range for both HIV and M.tuberculosis proteins, making this assay potentially useful in the clinical diagnosis of both HIV and Tuberculosis proteins indicative of active infection. Antigen detection for the HIV assay sensitivity was 89%, the specificity 85%. Serological detection had 100% sensitivity and specificity for the limited sample pool. The centrifugal microfluidic platform presented here offers the potential for a portable, fast and inexpensive multiplexed diagnostic device that can be used in resource-limited settings for diagnosis of TB and HIV.
The threats of disease outbreaks and exposure to biothreat agents, both accidental and intentional, demand field-deployable technology capable of rapid, sensitive, and accurate diagnosis. In order to address these public health concerns, we present a portable centrifugal microfluidic platform and demonstrate sensitive detection protein antigens, host response antibodies, and nucleic acids down to single digit starting copies. The nucleic acid detection utilizes an isothermal amplification via loop-mediated isothermal amplification (LAMP). The platform, which is composed of a compact optical system for laser induced fluorescence (LIF) detection, a quiet brushless motor, and an efficient non-contact heater, offers an easy-to-use system capable of performing sensitive biodetection in a constrained-resource environment.
Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.
Portable, sensitive, easy-to-use diagnostics are urgently needed to meet the growing need for advanced healthcare in the developing world. As the recent outbreaks of infectious diseases have demonstrated, early detection and treatment are vital tools to containing outbreaks and minimizing loss of life. Toward addressing these concerns, we have developed a centrifugal microfluidic platform capable of detecting both proteins and nucleic acids signatures from biological threats. This platform utilizes a novel sedimentation assay format to integrate sample preparation into a single step. Platform performance is competitive with traditional benchtop techniques.
Enteric and diarrheal diseases are a major cause of childhood illness and death in countries with developing economies. Each year, more than half of a million children under the age of five die from these diseases. We have developed a portable, microfluidic platform capable of simultaneous, multiplexed detection of several of the bacterial pathogens that cause these diseases. This platform can perform fast, sensitive immunoassays directly from relevant, complex clinical matrices such as stool without extensive sample cleanup or preparation. Using only 1 μL of sample per assay, we demonstrate simultaneous multiplexed detection of four bacterial pathogens implicated in diarrheal and enteric diseases in less than 20 min.
Waterborne pathogens pose significant threat to the global population and early detection plays an important role both in making drinking water safe, as well as in diagnostics and treatment of water-borne diseases. We present an innovative centrifugal sedimentation immunoassay platform for detection of bacterial pathogens in water. Our approach is based on binding of pathogens to antibody-functionalized capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk. Beads at the distal end of the disk are imaged to quantify the fluorescence and determine the bacterial concentration. Our platform is fast (20 min), can detect as few as ~10 bacteria with minimal sample preparation, and can detect multiple pathogens simultaneously. The platform was used to detect a panel of enteric bacteria (Escherichia coli, Salmonella typhimurium, Shigella, Listeria, and Campylobacter) spiked in tap and ground water samples.
The recent Ebola crisis in West Africa highlights challenges associated with pathogen diagnostics in the developing world, particularly logistical challenges with sample transport, availability of resources, and skilled labor. We present innovations in assay chemistry, microfluidic consumables, and smart phone-based instrumentation to enable a new generation of portable diagnostic devices.
The threats of disease outbreaks and bioterrorism demand field-deployable technology capable of rapid, sensitive, and accurate diagnosis. In order to address such public health concerns, we present a portable centrifugal microfluidic platform and demonstrate sensitive detection of E. coli down to single digit starting copies using isothermal amplification via loop-mediated isothermal amplification (LAMP). The platform, which is composed of a compact optical system for laser induced fluorescence (LIF) detection, a quiet brushless motor, and an efficient non-contact heater, offers an easy-to-use system capable of performing sensitive pathogen screening in a lab-free environment.
Koh, Chung-Yan K.; Schaff, Ulrich Y.; Piccini, Matthew E.; Stanker, Larry H.; Cheng, Luisa W.; Ravichandran, Easwaran; Singh, Bal R.; Sommer, Greg J.; Singh, Anup K.
We present an innovative centrifugal microfluidic immunoassay platform (SpinDx) to address the urgent biodefense and public health need for ultrasensitive point-of-care/incident detection of botulinum toxin. The simple, sample-to-answer centrifugal microfluidic immunoassay approach is based on binding of toxins to antibody-laden capture particles followed by sedimentation of the particles through a density-media in a microfluidic disk and quantification by laser-induced fluorescence. A blind, head-to-head comparison study of SpinDx versus the gold-standard mouse bioassay demonstrates 100-fold improvement in sensitivity (limit of detection = 0.09 pg/mL), while achieving total sample-to-answer time of <30 min with 2-∼L required volume of the unprocessed sample. We further demonstrate quantification of botulinum toxin in both exogeneous (human blood and serum spiked with toxins) and endogeneous (serum from mice intoxicated via oral, intranasal, and intravenous routes) samples. SpinDx can analyze, without any sample preparation, multiple sample types including whole blood, serum, and food. It is readily expandable to additional analytes as the assay reagents (i.e., the capture beads and detection antibodies) are disconnected from the disk architecture and the reader, facilitating rapid development of new assays. SpinDx can also serve as a general-purpose immunoassay platform applicable to diagnosis of other conditions and diseases.
Sandia journal manuscript; Not yet accepted for publication
Koh, Chung-Yan K.; Piccini, Matthew E.; Schaff, Ulrich, Y.; Stanker, Larry H.; Cheng, Luisa W.; Ravichandran, Easwaran R.; Singh, Bal-Ram S.; Sommer, Greg J.; Singh, Anup K.
Multiple cases of attempted bioterrorism events using biotoxins have highlighted the urgent need for tools capable of rapid screening of suspect samples in the field (e.g., mailroom and public events). We present a portable microfluidic device capable of analyzing environmental (e.g., white powder), food (e.g., milk) and clinical (e.g., blood) samples for multiplexed detection of biotoxins. The device is rapid (<15-30 min sample-to-answer), sensitive (< 0.08 pg/mL detection limit for botulinum toxin), multiplexed (up to 64 parallel assays) and capable of analyzing small volume samples (< 20 μL total sample input). The immunoassay approach (SpinDx) is based on binding of toxins in a sample to antibody-laden capture particles followed by sedimentation of particles through a density-media in a microfluidic disk and quantification using a laser-induced fluorescence detector. A direct, blinded comparison with a gold standard ELISA revealed a 5-fold more sensitive detection limit for botulinum toxin while requiring 250-fold less sample volume and a 30 minute assay time with a near unity correlation. A key advantage of the technique is its compatibility with a variety of sample matrices with no additional sample preparation required. Ultrasensitive quantification has been demonstrated from direct analysis of multiple clinical, environmental and food samples, including white powder, whole blood, saliva, salad dressing, whole milk, peanut butter, half and half, honey, and canned meat. We believe that this device can met an urgent need in screening both potentially exposed people as well as suspicious samples in mail-rooms, airports, public sporting venues and emergency rooms. The general-purpose immunodiagnostics device can also find applications in screening of infectious and systemic diseases or serve as a lab device for conducting rapid immunoassays.
Monitor i ng in f ectio n s in v ect o rs su c h as m osquit o es, s a nd fl i es, tsetse fl i es, a nd ticks to i denti f y hu m a n path o gens m a y s e r v e as a n ear l y w arn i ng det e ction system t o dir e ct loc a l g o v er n ment dise a se pr e v en t i v e m easu r e s . One major hurdle i n de t ection is the abi l i t y to scre e n l arge n u mbers of v e c t ors for h uman patho g ens w i thout t h e u s e of ge n o t y pe - s p ecific m o lecu l ar tec h nique s . N e x t genera t ion s equ e nc i ng (NG S ) pr o v i des a n unbi a sed p latfo r m capab l e of identi f y i ng k n o w n a n d unk n o w n p ath o ge n s circula t ing w i thin a v e ctor p opul a tion, but utili z ing t h is te c h nolo g y i s tim e - con s u ming a n d cos t l y for v ecto r -b o rne disease su r v e illan c e pr o gra m s. T o addr e s s this w e d e v e lop e d cos t -eff e ct i v e Ilumina(r) R NA- S eq l i bra r y p r epara t ion m e thodol o gies i n con j u n ction w i t h an automa t ed c ompu t at i onal a n a l y sis pipel i n e to ch a racter i ze t h e microbial popula t ions c ircula t i n g in Cu l e x m o squit o e s (Cul e x qui n quef a s c iatu s , C ul e x quinq u efasc i atus / pip i ens co m pl e x h y bri d s, and C u l e x ta r salis ) t hroug h out Californ i a. W e assembled 2 0 n o vel a n d w e l l -do c ume n ted a r b o v i ruses repres e nting mem b e rs of B u n y a v ir i da e , F l a v i virid a e, If a virida e , Meson i v i rida e , Nid o v iri d ae, O rtho m y x o virid a e, Pa r v o v iri d ae, Re o virid a e, R h a b d o v i rid a e, T y m o v iri d ae, a s w ell as s e v e r al u n assi g n e d v irus e s . In addit i o n, w e m app e d mRNA s pecies to d i vergent s peci e s of t r y panos o ma a nd pl a s modium eu k a r yotic parasit e s and cha r a c terized t he p r oka r yot i c microb i al c o mposit i on to i d enti f y bacteri a l tran s c r ipts der i v ed from wolba c hia, clo s tridi u m, m y c oplas m a, fusoba c terium and c am p y l o bacter bac t er i al spec i e s . W e utilized the s e mic r obial transcri p tomes pre s e nt in g e ogra p hical l y defined Cul e x po p ul a tions to defi n e spatial and m osqui t o specie s -spec i fic ba r r iers of i n fecti o n. T he v i r ome and microbi o me c o mpos i tion id e ntified in e ach mosqui t o p o ol pr o v i ded suf f icient resolut i on to dete r m i ne both the mosq u ito species and the g e o graphic regi o n in Californ i a w h e re t h e mosqui t o po o l orig i n ated. T his d a ta pr o v i des ins i ght in t o the compl e x i t y of microb i al spec i es cir c ulati n g in med i cal l y i mport a nt Culex mosqui t oes a nd t h eir potent i al im p act o n t he tran s missi o n of v ector-b o rne human / veter i na r y p a t hogens in C a liforn i a.