Yi Xiao

Abstract

NAD(P)H and its oxidized form, NAD(P) + are ubiquitous biomolecules associated with cellular energy metabolism in both eukaryotic and prokaryotic organisms. It has been reported that the NAD(P)+/NAD(P)H couples are essential cofactors for more than 300 dehydrogenases. Increased activity of dehydrogenases, such as aldehyde dehydrogenases, has been reported in various human cancers and has been found to interfere with certain chemotherapeutic treatments. Several dehydrogenase inhibitors have been developed for the treatment of human diseases as well as applications in alcohol dependence, cocaine addiction, anxiety, and as resensitizing agents for cancers. Traditionally, colorimetric detection of NADH is based on the growth of gold nanoparticles, which requires a large sample volume, a longer reaction time, as well as sophisticated analytical instruments to confirm detection efficacy. In addition, it is hard to achieve a low detection limit due to the interference arising from background absorbance.FIU inventors have developed diagnostic devices and methods that require less sample volume and easier method of fabrication, while maintaining the sensitivity for detecting analytes critical for biological activities in various applications. These devices include a paper-based sensor that takes advantage of the NADH-inhibited dissolution of gold nanoparticles to significantly reduce the background and thereby lower the detection limit. The sensor surface-confined testing zone minimizes sample volume, and capillary force-assisted vertical diffusion improves the efficiency of mass transportation. As a result, the sensor requires only 4 minutes at room temperature to analyze a 25 μL sample with a detection limit of 12.5 μM NADH in 20% cell lysate by naked-eye examination.

Benefit

Instrument free Easy colorimetric readout Rapid and sensitive room-temperature detection Requires small sample volume Low-cost

Market Application

Naked-eye monitoring of NAD+-mediated enzymatic reactionsIdentification of dehydrogenase inhibitors for the development of novel anticancer agents, antibiotics, and pesticides

Publications

Paper-Based Device for Rapid Visualization of NADH Based on Dissolution of Gold Nanoparticles, ACS Appl. Mater. Interfaces 2015, 7, 27, 15023–15030

Abstract

FIU inventors have created a new approach for preparing paper-based gold films with excellent conductive and electrochemical properties. A simple, ambient vacuum filtration method is used to rapidly generate gold films without the need for sophisticated instruments or a clean-room environment. The process takes 25 minutes and requires only two steps of filtration with two aqueous solutions. This is the first report on the use of metallic single-walled carbon nanotubes (M-SWCNTs) as an interface for the direct deposition of AuNPs to immediately form a conductive, three-dimensional gold film on a paper substrate. Easy to use, economical, and environmentally friendly mixed cellulose ester (MCE) filter paper supports the hybrid nanostructure. The resulting 3D porous gold films demonstrate a high surface area-to-volume ratios, high conductivity, and good mechanical properties. This procedure offers a promising new approach that could be readily generalized for the highly reproducible fabrication of various metal films at ambient conditions, and could also be used as a prelude to transferring the resulting film to other metal or flexible substrates.

Benefit

Porous film offers a superior electrode material for electrocatalytic detection relative to standard flat gold slidesGenerates a larger oxidation current for electroactive molecules such as dopamine and serotoninCapable of discriminating the simultaneous presence of both moleculesMCE filter paper is simple, low-cost, biodegradable, combustible, portable and eco-friendly

Market Application

Electrochemical catalysisElectro-optical devicesReflective, conductive or energy-collecting metallic coatings

Publications

ACS Appl. Mater. Interfaces 2015, 7, 49, 27049–27058ACS Appl Mater Interfaces. 2021 Apr 21; 13(15): 17330–17339

Abstract

Antibody-based screens often suffer from poor specificity, and existing aptamer-based detection generally rely on polymerase amplification, enzymes or enzyme-linked assays to achieve sensitive cocaine detection at low nanomolar concentrations. Preparation for these multi-step assays is complex and usually requires at least several hours.FIU inventors have created a rapid and specific target-displaced, aptamer-based method for one-step cocaine detection with minimal reagent requirement. The discovery of the interaction between the ATMND molecule and the cocaine-binding aptamer, 38-GC, has called for the design of a unique sensor. The new sensor platform relies on the displacement of ATMND from the 38-GC aptamer by cocaine through competitive binding. The 38-GC aptamer was engineered through the introduction of sequence changes into the MNS-4.1 and exhibits higher affinity to both ligands while reducing background signals and increasing signal gain. Through successful testing, the sensor has demonstrated detection of cocaine within seconds at concentrations as low as 200 nM, which is 50-fold lower than the assays based on target-induced conformational change. Moreover, signal gain reportedly increased with the increase of cocaine concentration, reaching a limit of detection of 10.4 µM, 18.4 µM, and 36 µM in undiluted saliva, urine, and serum samples respectively. The label-free detection is performed at room temperature in a single tube containing the aptamer-ATMND complex and the sample of interest.

Benefit

Able to detect cocaine within seconds at concentrations as low as 200 nMOne-step, label-free method of preparation is quick and simpleAptamer-based detection results in high specificity

Market Application

This approach offers a general aptamer-based framework for sensitive, specific and high-throughput on-site drug testing.

Abstract

Fentanyl and its analogs are highly potent synthetic opioids. Fentanyl is used to treat patients with severe pain or to manage pain after surgery. Unfortunately, it can produce drug dependence and tolerance which can lead to significant issues. Current methods for fentanyl/fentanyl analogs screening are prone to false negatives and positives, and a need remains to develop new tools to rapidly and selectively detect these small molecules.Aptamers are nucleic acid-based or peptides that are isolated from randomized nucleic-acid or peptides libraries. Their low cost, ease of modification, and long shelf life make them good candidates as bioreceptors for biosensing, imaging, and therapeutic applications. Moreover, aptamers have a cross-reactivity functionality that can be tuned to target a specific core structure and increase specificity against non-family compounds. Therefore, aptamers are an excellent alternative as bioreceptors for the detection of small molecules that share the same core structure, such as fentanyl and its analogs.FIU scientists have developed aptamers and aptamer-based sensors for the detection of fentanyl and its analogs in body fluids and seized samples. The aptamers bind to these small molecules with nanomolar affinity and high specificity against illicit drugs, adulterants, and cutting agents commonly existing in seized samples. The aptamers have been used in single-step fluorescence and electrochemical sensors

Benefit

Rapid detection of target compounds (withing second)Compatible for both clinical and field settingsLow nanomolar-micromolar affinity for the target compounds Minimal response to non-target compounds

Market Application

Drug detection/screeningLaw enforcement Medical diagnosticsEnvironmental monitoringFood safety

Abstract

The biosensor field is continuously developing methods to achieve greater sensitivity. The sensitivity of a biosensor is determined by the intrinsic target affinity of the bioreceptor being used for detection. Highly sensitive methods such as high-performance liquid chromatography have been used for the detection of small molecules. However, these methods are time-consuming and require expensive reagents and equipment as well as complex sample preparation. Aptamers are nucleic acid-based affinity reagents that can be isolated for any target, are chemically stable, and can be synthesized at a low cost with high reproducibility. However, most aptamers do not have inherent structure-switching functionality, and a multi step process is required to introduce this functionality for sensor development. Current methods are labor-intensive and require considerable trial and error. Moreover, engineered structure switching aptamers often have low target-binding affinities, greatly limiting their utility. FIU scientists have developed methods for generating structure-switching aptamers from fully folded or pre-folded aptamers. The generated aptamers can be modified with a fluorophore-quencher pair, and the detection of the small molecule target is measured by a signal generated upon binding of the small-molecule target to the aptamer-based sensor. The generated aptamers can also be incorporated into an electrochemical aptamer-based (E-AB) sensor and achieves excellent sensor performance. The technology has been successfully tested with DIS, cocaine, ATP, and synthetic cathinones.

Benefit

Rapidly introduce structure-switching functionality into small-molecule-binding aptamersEffective detection of small-molecule targets in a complex sample (single pot multiplex detection)Only require a single low-volume of sampleCan accurately determine the concentration of the targets

Market Application

medical diagnostics and monitoring Drug screeningEnvironmental monitoringBiodefenseFood safetyLaw enforcementPublic health

Abstract

Synthetic cathinones, also known as 'bath salts', are designer drugs sharing a similar core structure with amphetamines and 3,4-methylenedioxy-methamphetamine (MDMA). They are highly addictive central nervous system stimulants that are associated with many negative health consequences, and can even cause death. Although these drugs have only emerged recently, abuse of bath salts has become a threat to public health and safety due to their severe toxicity, increasingly broad availability, and difficulty of regulation. More importantly, there is currently no reliable presumptive test for any synthetic cathinones. Chemical spot tests used to detect conventional drugs such as cocaine, methamphetamine, and opioids show no cross-reactivity to synthetic cathinones.FIU inventors have developed a DNA aptamer isolated from systematic evolution of ligands by exponential enrichment (SELEX) binding to 3,4-methylenedioxypyrovalerone (MDPV) and numerous members of the synthetic cathinone family. The isolated MDPV-binding aptamer showed high cross-reactivity to other synthetic cathinone analogs, including naphyrone, methylone, pentylone, and mephedrone, but not for structurally-similar drugs, like amphetamine and methamphetamine, and common cutting agents, such as lidocaine, benzocaine, and caffeine. Once the synthetic cathinone binds to the aptamer-based sensor, a change in absorbance or fluorescence intensity is observed. The absorbance read-out can be quantified by, for example, a microplate-reader or portable photometer, allowing for high-throughput detection.

Benefit

Detects and quantifies MDPV and other synthetic cathinone analogs at concentrations as low as 200 ng/mL within secondsHigh specificity against cutting agents and structurally-similar non-cathinone compounds interferences

Market Application

Drug testing in clinical and field settings

Publications

Haixiang Yu, Weijuan Yang, Obtin Alkhamis, Juan Canoura, Kyung-Ae Yang, Yi Xiao, In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality, Nucleic Acids Research, Volume 46, Issue 8, 4 May 2018, Page e43, https://doi.org/10.1093/nar/gky026

Abstract

Across the country states are legalizing marijuana for medical and/or recreational uses. As the legal landscape is rapidly changing, there are growing concerns about marijuana impairment while driving or in the workplace, and an increasing demand by law enforcement personnel for the capability to differentiate industrial hemp crops from recreational marijuana. Quantification of cannabinoids in human biological samples and plant material is currently performed in centralized laboratory settings. However, there are no reliable tests that can be employed on-site for the determination of cannabinoid content in a variety of sample matrices.FIU inventors have developed aptamers-based sensors and methods for the detection of cannabinoids in body fluids. One aptamer specifically binds to tetrahydrocannabinol (THC) with nanomolar dissociation constant and does not bind to structurally similar cannabinoids such as cannabidiol and cannabinol. This aptamer also binds to the urinary metabolite of THC, THC-COOH. The second aptamer binds to the synthetic cannabinoids UR-144 and XLR-11, each with nanomolar dissociation constants, and does not bind to any naturally occurring cannabinoids.

Benefit

Achieves instantaneous detections of as low as 1 µg/mL THC and THC-COOH with the naked eye and 70 ng/mL THC and THC-COOH via fluorescence detectionSimultaneous detection of THC and its metabolites enables onsite detection of recent cannabis use in both urine and saliva samplesExcellent specificity, no response to endogenous cannabinoids in cannabis plants and commonly used illicit drugs

Market Application

Drug screening in both clinical and field settings such as workplaces, drug testing laboratories, Criminal justice systems, law enforcement agencies, hospitals, drug treatment centers, individual users, pain management centers, schools & colleges

Abstract

Small molecules are important targets with the potential of clinical or commercial applications such as medical diagnostics, environmental monitoring, and forensic science. Thus, efforts to develop methods for portable, low-cost, and quantitative on-site detection of a broad range of small molecules are gaining momentum.Various immunoassays have also been developed for the detection of small molecules such as cocaine and/or its major metabolite benzoylecgonine in biofluids, including the enzyme-linked immunosorbent assay (ELISA). Unfortunately, the use of immunoassays for the detection of designer drugs is often limited because of the high cost of generating new antibodies and issues with narrow target binding-spectrum and poor specificity, and most of these assays offer only limited capabilities for naked-eye detection, because the resulting absorbance changes can only be detected by instruments. FIU inventors have developed aptamer sensors that report the presence of small-molecule target such as cocaine and synthetic cathinones via a sensitive colorimetric signal for naked-eye detection. The aptamer sensors are CBSAzyme-based sensors having both target-mediated cooperative behavior of the CBSA and peroxidase-mimicking catalytic activity of DNAzyme. The CBSAzyme-based sensors comprise a long fragment and a short fragment, the long fragment comprising a first segment of a split DNAzyme and a long fragment of a CBSA, the short fragment comprising a second segment of the split DNAzyme and a short fragment of the CBSA. The two fragments of the CBSAzyme remain separate in the absence of the small-molecule target, but effectively assemble in the presence of the small-molecule target. The assembly of the two fragments of the CBSAzyme activates the DNAzyme that subsequently catalyzes the oxidation of 2,2'-azinobis(3-ethylbenzthiazo-line)-6-sulfonic acid (ABTS), producing a visible color change from colorless to dark green that reveals the presence of the target within minutes.In an initial demonstration, the inventors have generated a cocaine-binding CBSAzyme that enables naked-eye detection of this drug at concentrations as low as 10 uM within 15 minutes. They subsequently demonstrated the generality of this assay strategy by coupling the same split DNAzyme into a CBSAzyme that responds to the designer drug methylenedioxypyrovalerone (MDPV). This CBSAzyme enabled visual detection of MDPV and 11 other synthetic cathinones at concentrations as low as 30 uM within 15 minutes, but did not respond to five common cutting agents.

Benefit

Naked-eye detection of small molecules within minutesHigh sensitivity and specificityCompatible for both clinical and field settingsLabel-free and instrument-free

Market Application

Colorimetric assays in both clinical and field settings for small-molecule targets including illicit drugs, toxins, disease biomarkers, and pharmaceuticals for applications such as law enforcement, environmental monitoring, and clinical diagnostics.

Publications

Luo Y, Yu H, Alkhamis O, et al. Label-Free, Visual Detection of Small Molecules Using Highly Target-Responsive Multimodule Split Aptamer Constructs. Anal Chem. 2019;91(11):7199-7207. doi:10.1021/acs.analchem.9b00507

Abstract

Enzyme-assisted target recycling (EATR) is an effective way to amplify signals generated from target-binding events. With this technique, a substantial fluorescent signal can be generated by a single copy of the target, thereby greatly decreasing the limit of detection. Aptamers are nucleic acid-based bio-recognition elements that can bind to a wide variety of targets, including proteins, metal ions, small molecules, and even whole cells. Many strategies have been employed in aptamer-based assays to achieve target detection in an instrument-free manner. In particular, aptamers can be split into two or three fragments that remain separate in the absence of target but assemble upon target binding. In principle, the target-induced assembly of split aptamers should be compatible with EATR-mediated signal amplification. However, it is difficult to achieve sensitive target induced aptamer assembly with conventional split aptamers with a single binding-domain. FIU inventors have developed a CBSA-based EATR-amplified fluorescence assay to quantify dehydroisoandrosterone-3-sulfate (DIS) in urine, which is a diagnostic biomarker for adrenal tumors. This assay achieved 100-fold enhanced target sensitivity relative to a non-EATR-based assay, with a detection limit of 1 μM in 50% urine within 30 minutes.

Benefit

Cost effectiveRapid, sensitive, and specificNaked-eye or fluorescent detection

Market Application

Adrenal cancer test

Abstract

Synthetic cathinones (also known as bath salts) are designer drugs sharing a similar core structure with amphetamines and 3, 4-methylenedioxy-methamphetamine (MDMA). They are highly addictive central nervous system stimulants, and are associated with many negative health consequences, including even death. Although these drugs have emerged only recently, abuse of bath salts has become a threat to public health and safety due to their severe toxicity, increasingly broad availability, and difficulty of regulation. More importantly, there is currently no reliable presumptive test for any synthetic cathinone. Chemical spot tests used to detect conventional drugs such as cocaine, methamphetamine, and opioids show no cross-reactivity to synthetic cathinones. FIU inventors have developed cross-reactive aptamers with structure-switching functionality and sensors for the detection of synthetic cathinones. The generation of a structure-switching cross-reactive aptamer entails digesting the cross-reactive aptamer with an exonuclease mixture, such as exonuclease III (Exo Ill) and exonuclease I (Exo I). The resulting digestion product has structure-switching functionality with similar or equal affinity as its parent cross-reactive aptamer and can be directly employed in folding-based aptamer sensors. The structure-switching cross-reactive aptamers have been used in two different type of assays. One is a label free dye displacement assay where the presence of synthetic cathinones is reported via a sensitive colorimetric signal for naked-eye detection. This colorimetric assay has excellent specificity because the aptamer does not cross-react to non-synthetic cathinone interferents, can detect nanomolar synthetic cathinone concentrations even in urine and saliva in a label-free manner via instrumental means, and can also achieve instantaneous detection of as low as 6.3 μM with the naked-eye when the dye is used at a micromolar concentration. The second assay consist of incorporating the structure-switching cross-reactive aptamers into an electrochemical aptamer-based (E-AB) sensor, which comprises an electrode, and the detection of the synthetic cathinone(s) is done by measuring an increase in current generated upon binding of the synthetic cathinone(s) to the sensor. E-AB sensors are an ideal choice for the detection of seized substances, because they are insensitive to sample matrix effects, have rapid response times (seconds-scale), can perform detection with minimal sample preparation requirements, and can be easily miniaturized to detect low-volume samples (micro liters), thus allowing for analysis of trace amounts of substances.

Benefit

1. Colorimetric detection:Compatible for both clinical and field settingsColor change from colorless to bright blue within minutesCan be performed in a single tubeHas detection limit suitable for screening in biosamples2. Electrochemical detection:High sensitivity and specificityRead-out can be quantified in secondsInsensitive to sample matrix effectsMinimal sample preparation requirements

Market Application

Drug testing in clinical and field settings