Portable and Label-Free Sensor Array for Discriminating Multiple Analytes via a Handheld Gas Pressure Meter.

Cross-reactive sensor arrays are useful for discriminating multiple analytes in a complex sample. Herein, a portable and label-free gas pressure sensor array was proposed for multiplex analysis via a handheld gas pressure meter. It is based on the interaction diversity of analytes with catalase-like nanomaterials, including Pt nanoparticles (PtNP), Co3O4 nanosheets (Co3O4NS), and Pt-Co alloy nanosheets (PtCoNS), respectively. Thus, the diverse influence of analytes on the catalase-like activity could be output as the difference in the gas pressure. By using principal component analysis, eight proteins were well distinguished by the gas pressure sensor array at the 10 nM level within 12 min. Moreover, different concentrations of proteins and mixtures of proteins could likewise be discriminated. More importantly, the effective discrimination of proteins in human serum and discrimination of five kinds of cells further confirmed the potential of the gas pressure sensor array. Therefore, it provides a portable, cheap, sensitive, and label-free gas pressure sensor array, which is totally different from the reported sensor arrays and holds great potential for portable and cheap discrimination of multiple analytes.

A polypyrrole-mediated photothermal biosensor with a temperature and pressure dual readout for the detection of protein biomarkers.

Photothermal biosensors with advantages of speed and high sensitivity offer alternative and reliable solutions for real-time clinical diagnosis, food safety testing and environmental monitoring. Although metallic nanoparticles are usually used for photothermal biosensors, their poor photothermal stability and potential toxicity hinder clinical applications. Taking advantage of the low cytotoxicity and remarkable photothermal effect under the low laser power of polypyrrole-based organic nanoparticles, we developed a novel photothermal biosensor with a temperature and pressure dual readout. After the formation of immunoassay sandwich structures, polypyrrole as the photothermal agent is synthesized in situ with pyrrole, HCl and Fe3+ released from magnetic Fe2O3 particles modified with detection antibody. The heterocyclic rings from polypyrrole enable photothermal performance in the NIR region. The resulting increased heat and pressure in a sealed well are measured using a digital thermometer and a portable pressure meter, respectively. Taking C-reactive protein (CRP) as a model target, the proposed strategy allowed sensitive, selective and accurate analysis of biomarkers, and showed performance comparable to that of ELISA. Overall, the dual-mode photothermal biosensor holds great potential for simple and low-cost photothermal sensing of biomarkers for point-of-care testing (POCT).

An electrochemical method for a rapid and sensitive immunoassay on digital microfluidics with integrated indium tin oxide electrodes coated on a PET film.

Electrochemical detection is the simplest analytical tool to be integrated into digital microfluidics (DMF). It offers the advantages of small size, with detector electrodes incorporated into the device by patterning, and high compatibility with portable analytical instruments. Indium tin oxide (ITO) coated on glass has been commonly used for the top plate of DMF due to its good conductivity and transparency. However, instability and the low current response of ITO electrodes patterned on glass hindered their application for immunoassays. It has been reported that ITO coated on polyethylene terephthalate (PET) has better conductivity, owing to its higher carrier concentration, faster mobility and lower resistivity. Herein, we investigated the use of ITO electrodes patterned on PET film as the top plate of DMF for a simple and stable electrochemical immunoassay using square wave voltammetry (SWV), with an excellent peak resolution and high sensitivity. A magnetic bead-based immunoassay for H5N1 antigen was performed on a DMF platform with a limit of detection of 0.6 ng mL-1 in buffer and 18 ng mL-1 in human serum. These results showed the good electrochemical performance of ITO coated on a PET film, a lightweight, shock resistant and cost-effective material, which is promising for DMF fabrication and transparent electrodes for various electroanalytical methods.

Microfluidic Distance Readout Sweet Hydrogel Integrated Paper-Based Analytical Device (µDiSH-PAD) for Visual Quantitative Point-of-Care Testing.

A disposable, equipment-free, versatile point-of-care testing platform, microfluidic distance readout sweet hydrogel integrated paper-based analytical device (µDiSH-PAD), was developed for portable quantitative detection of different types of targets. The platform relies on a target-responsive aptamer cross-linked hydrogel for target recognition, cascade enzymatic reactions for signal amplification, and microfluidic paper-based analytic devices (µPADs) for visual distance-based quantitative readout. A "sweet" hydrogel with trapped glucoamylase (GA) was synthesized using an aptamer as a cross-linker. When target is present in the sample, the "sweet" hydrogel collapses and releases enzyme GA into the sample, generating glucose by amylolysis. A hydrophilic channel on the µPADs is modified with glucose oxidase (GOx) and colorless 3,3'-diaminobenzidine (DAB) as the substrate. When glucose travels along the channel by capillary action, it is converted to H2O2 by GOx. In addition, DAB is converted into brown insoluble poly-3,3'-diaminobenzidine [poly(DAB)] by horseradish peroxidase, producing a visible brown bar, whose length is positively correlated to the concentration of targets. The distance-based visual quantitative platform can detect cocaine in urine with high selectivity, sensitivity, and accuracy. Because the target-induced cascade reaction is triggered by aptamer/target recognition, this method is widely suitable for different kinds of targets. With the advantages of low cost, ease of operation, general applicability, and disposability with quantitative readout, the µDiSH-PAD holds great potential for portable detection of trace targets in environmental monitoring, security inspection, personalized healthcare, and clinical diagnostics.

Surface-Enhanced Raman Scattering Active Plasmonic Nanoparticles with Ultrasmall Interior Nanogap for Multiplex Quantitative Detection and Cancer Cell Imaging.

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

Target-responsive DNA hydrogel mediated "stop-flow" microfluidic paper-based analytic device for rapid, portable and visual detection of multiple targets.

A versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic paper-based analytic device (µPAD) using a target-responsive hydrogel to mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel was used as a target-responsive flow regulator in the µPAD. In the absence of a target, the hydrogel is formed in the flow channel, stopping the flow in the µPAD and preventing the colored indicator from traveling to the final observation spot, thus yielding a "signal off" readout. In contrast, in the presence of a target, no hydrogel is formed because of the preferential interaction of target and aptamer. This allows free fluidic flow in the µPAD, carrying the indicator to the observation spot and producing a "signal on" readout. The device is inexpensive to fabricate, easy to use, and disposable after detection. Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment. Multiple targets, including cocaine, adenosine, and Pb(2+), can be detected simultaneously, even in complex biological matrices such as urine. The reported method offers simple, low cost, rapid, user-friendly, point-of-care testing, which will be useful in many applications.

Simple and Rapid Functionalization of Gold Nanorods with Oligonucleotides Using an mPEG-SH/Tween 20-Assisted Approach.

DNA conjugated gold nanorods (AuNRs) are widely applied for nanostructure assembly, gene therapy, biosensing, and drug delivery. However, it is still a great challenge to attach thiolated DNA on AuNRs, because the positively charged AuNRs readily aggregate in the presence of negatively charged DNA. This article reports an mPEG-SH/Tween 20-assisted method to load thiolated DNA on AuNRs in 1 h. Tween 20 and mPEG-SH are used to synergistically displace CTAB on the surface of AuNRs by repeated centrifugation and resuspension, and thiolated DNA are attached to AuNRs in the presence of 1 M NaCl, 100 mM MgCl2, or 100 mM citrate. AuNRs with different sizes and aspect ratios can be functionalized with DNA by this method. The number of DNA loaded on each AuNR can be easily controlled by the concentrations of mPEG-SH and Tween 20 or the ratio between DNA and AuNR. Functionalized AuNRs were used for nanoparticle assembly and cancer cell imaging to confirm that DNA anchored on the surface of AuNRs retains its hybridization and molecular recognition capability. The new method is easy, rapid, and robust for the preparation of DNA functionalized AuNRs for a variety of applications such as cancer therapy, drug delivery, self-assembly, and imaging.

Target-responsive DNAzyme cross-linked hydrogel for visual quantitative detection of lead.

Because of the severe health risks associated with lead pollution, rapid, sensitive, and portable detection of low levels of Pb(2+) in biological and environmental samples is of great importance. In this work, a Pb(2+)-responsive hydrogel was prepared using a DNAzyme and its substrate as cross-linker for rapid, sensitive, portable, and quantitative detection of Pb(2+). Gold nanoparticles (AuNPs) were first encapsulated in the hydrogel as an indicator for colorimetric analysis. In the absence of lead, the DNAzyme is inactive, and the substrate cross-linker maintains the hydrogel in the gel form. In contrast, the presence of lead activates the DNAzyme to cleave the substrate, decreasing the cross-linking density of the hydrogel and resulting in dissolution of the hydrogel and release of AuNPs for visual detection. As low as 10 nM Pb(2+) can be detected by the naked eye. Furthermore, to realize quantitative visual detection, a volumetric bar-chart chip (V-chip) was used for quantitative readout of the hydrogel system by replacing AuNPs with gold-platinum core-shell nanoparticles (Au@PtNPs). The Au@PtNPs released from the hydrogel upon target activation can efficiently catalyze the decomposition of H2O2 to generate a large volume of O2. The gas pressure moves an ink bar in the V-chip for portable visual quantitative detection of lead with a detection limit less than 5 nM. The device was able to detect lead in digested blood with excellent accuracy. The method developed can be used for portable lead quantitation in many applications. Furthermore, the method can be further extended to portable visual quantitative detection of a variety of targets by replacing the lead-responsive DNAzyme with other DNAzymes.

Au@Pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing.

Point-of-care testing (POCT) with the advantages of speed, simplicity, portability, and low cost is critical for the measurement of analytes in a variety of environments where access to laboratory infrastructure is lacking. While qualitative POCTs are widely available, quantitative POCTs present significant challenges. Here we describe a novel method that integrates an Au core/Pt shell nanoparticle (Au@PtNP) encapsulated target-responsive hydrogel with a volumetric bar-chart chip (V-Chip) for quantitative POCT. Upon target introduction, the hydrogel immediately dissolves and releases Au@PtNPs, which can efficiently catalyze the decomposition of H2 O2 to generate a large volume of O2 to move of an ink bar in the V-Chip. The concentration of the target introduced can be visually quantified by reading the traveling distance of the ink bar. This method has the potential to be used for portable and quantitative detection of a wide range of targets without any external instrument.

Target-responsive "sweet" hydrogel with glucometer readout for portable and quantitative detection of non-glucose targets.

Portable devices with the advantages of rapid, on-site, user-friendly, and cost-effective assessment are widely applied in daily life. However, only a limited number of quantitative portable devices are commercially available, among which the personal glucose meter (PGM) is the most successful example and has been the most widely used. However, PGMs can detect only blood glucose as the unique target. Here we describe a novel design that combines a glucoamylase-trapped aptamer-cross-linked hydrogel with a PGM for portable and quantitative detection of non-glucose targets. Upon target introduction, the hydrogel collapses to release glucoamylase, which catalyzes the hydrolysis of amylose to produce a large amount of glucose for quantitative readout by the PGM. With the advantages of low cost, rapidity, portability, and ease of use, the method reported here has the potential to be used by the public for portable and quantitative detection of a wide range of non-glucose targets.

Preparation of reversible colorimetric temperature nanosensors and their application in quantitative two-dimensional thermo-imaging.

Reversible colorimetric temperature nanosensors were prepared using a very simple precipitation method to encapsulate two color luminescent dyes. These nanosensors presented obvious reversible temperature response and enabled both rapid colorimetric temperature estimation using the eyes and quantitative two-dimensional thermo-imaging. Heat-exchange induced fluid motion was, for the first time, rapidly, precisely, and quantitatively imaged by just taking color pictures, and this presented good temporal and spatial resolution for studying heat-driven hydrodynamics. These nanosensors should have great application in micro/nanoscale research and also fabrication into films for macroscopic study.

Distance-based paper/PMMA integrated ELISA-chip for quantitative detection of immunoglobulin G.

The enzyme-linked immunosorbent assay (ELISA) is one of the most commonly implemented clinical diagnostic tools for the detection and quantification of protein biomarkers. However, conventional ELISA tests require sophisticated infrastructure, expensive reagents, long assay time, and expertise for operation, which are not often easily accessible in resource-limited settings. Microfluidic ELISA-chip based point-of-care (POC) testing allows miniaturization and integration of complex functions that facilitate their usage in limited-resource settings. The current work demonstrates a simple, portable, low cost and equipment-free paper/poly(methyl methacrylate) (PMMA) integrated microfluidic ELISA-chip as a POC device with a visual distance-based readout for quantitative detection of clinical biomarkers. The integrated paper/PMMA ELISA-chip utilizes the movement of immunoassay complexes with magnetic beads by a permanent magnet in a PMMA part of the compartment. The target concentration is translated into a visual distance signal readout for quantitative detection of biomarkers in a µPAD. Because it does not require sophisticated instruments and has the added advantages of low cost, easy operation, and disposability with quantitative visual readout, the paper/PMMA ELISA-chip holds great promise for portable detection of target bioanalytes as a POC diagnostic tool in resource-limited setups.

PMMA/PDMS valves and pumps for disposable microfluidics.

Poly(methyl methacrylate) (PMMA) is gaining in popularity in microfluidic devices because of its low cost, excellent optical transparency, attractive mechanical/chemical properties, and simple fabrication procedures. It has been used to fabricate micromixers, PCR reactors, CE and many other microdevices. Here we present the design, fabrication, characterization and application of pneumatic microvalves and micropumps based on PMMA. Valves and pumps are fabricated by sandwiching a PDMS membrane between PMMA fluidic channel and manifold wafers. Valve closing or opening can be controlled by adjusting the pressure in a displacement chamber on the pneumatic layer via a computer regulated solenoid. The valve provides up to 15.4 microL s(-1) at 60 kPa fluid pressure and seals reliably against forward fluid pressure as high as 60 kPa. A PMMA diaphragm pump can be assembled by simply connecting three valves in series. By varying valve volume or opening time, pumping rates ranging from nL to microL per second can be accurately achieved. The PMMA based valves and pumps were further tested in a disposable automatic nucleic acid extraction microchip to extract DNA from human whole blood. The DNA extraction efficiency was about 25% and the 260 nm/280 nm UV absorption ratio for extracted DNA was 1.72. Because of its advantages of inexpensive, facile fabrication, robust and easy integration, the PMMA valve and pump will find their wide application for fluidic manipulation in portable and disposable microfluidic devices.

High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets.

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation. After bulk PCR amplification, the droplets are lysed and the beads are recovered and rapidly analyzed via flow cytometry. DNA targets ranging in size from 380 to 1139 bp at single molecule concentrations are quantitatively amplified using SCGA. Long-range sequencing results from beads each carrying approximately 100 amol of a 624 bp product demonstrate that these amplicons are competent for achieving attomole-scale Sanger sequencing from a single bead and for advancing pyrosequencing read-lengths. Successful single cell analysis of the glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene in human lymphocyte cells and of the gyr B gene in bacterial Escherichia coli K12 cells establishes that SCGA will also be valuable for performing high-throughput genetic analysis on single cells.

Investigation of the hybrid molecular probe for intracellular studies.

Monitoring gene expression in vivo is essential to the advancement of biological studies, medical diagnostics, and drug discovery. Adding to major efforts in developing molecular probes for mRNA monitoring, we have recently developed an alternative tool, the hybrid molecular probe (HMP). To optimize the probe, a series of experiments were performed to study the properties of HMP hybridization kinetics and stability. The results demonstrated the potential of the HMP as a prospective tool for use in both hybridization studies and in vitro and in vivo analyses. The HMP has shown no tendency to produce false positive signals, which is a major concern for living cell studies. Moreover, HMP has shown the ability to detect the mRNA expression of different genes inside single cells from both basal and stimulated genes. As an effective alternative to conventional molecular probes, the proven sensitivity, simplicity, and stability of HMPs show promise for their use in monitoring mRNA expression in living cells.

A portable visual detection method based on a target-responsive DNA hydrogel and color change of gold nanorods.

A new method based on a functional DNA crosslinked hydrogel as a target-responsive unit and gold nanorods (AuNRs) as a multicolor signal readout circuit was developed for the sensitive and visual detection of different targets. The color variation of the AuNR solution was correlated with the concentration of the target. This system can be extended to detect various targets by designing the corresponding target-responsive DNA hydrogels.

Integrating Target-Responsive Hydrogel with Pressuremeter Readout Enables Simple, Sensitive, User-Friendly, Quantitative Point-of-Care Testing.

Point-of-care testing (POCT) with the advantages of speed, simplicity, and low cost, as well as no need for instrumentation, is critical for the measurement of analytes in a variety of environments lacking access to laboratory infrastructure. In the present study, a hydrogel pressure-based assay for quantitative POCT was developed by integrating a target-responsive hydrogel with pressuremeter readout. The target-responsive hydrogels were constructed with DNA grafted linear polyacrylamide and the cross-linking DNA for selective target recognition. The hydrogel response to the target substance allows release of the preloaded Pt nanoparticles, which have good stability and excellent catalytic ability for decomposing H2O2 to O2. Then, the generated O2 in a sealed environment leads to significant pressure increase, which can be easily read out by a handheld pressuremeter. Using this target-responsive hydrogel pressure-based assay, portable and highly sensitive detection of cocaine, ochratoxin A, and lead ion were achieved with excellent accuracy and selectivity. With the advantages of portability, high sensitivity, and simple sample processing, the hydrogel pressure-based assay shows great potential for quantitative POCT of a broad range of targets in resource-limited settings.

Design and synthesis of target-responsive hydrogel for portable visual quantitative detection of uranium with a microfluidic distance-based readout device.

Due to uranium's increasing exploitation in nuclear energy and its toxicity to human health, it is of great significance to detect uranium contamination. In particular, development of a rapid, sensitive and portable method is important for personal health care for those who frequently come into contact with uranium ore mining or who investigate leaks at nuclear power plants. The most stable form of uranium in water is uranyl ion (UO2(2+)). In this work, a UO2(2+) responsive smart hydrogel was designed and synthesized for rapid, portable, sensitive detection of UO2(2+). A UO2(2+) dependent DNAzyme complex composed of substrate strand and enzyme strand was utilized to crosslink DNA-grafted polyacrylamide chains to form a DNA hydrogel. Colorimetric analysis was achieved by encapsulating gold nanoparticles (AuNPs) in the DNAzyme-crosslinked hydrogel to indicate the concentration of UO2(2+). Without UO2(2+), the enzyme strand is not active. The presence of UO2(2+) in the sample activates the enzyme strand and triggers the cleavage of the substrate strand from the enzyme strand, thereby decreasing the density of crosslinkers and destabilizing the hydrogel, which then releases the encapsulated AuNPs. As low as 100nM UO2(2+) was visually detected by the naked eye. The target-responsive hydrogel was also demonstrated to be applicable in natural water spiked with UO2(2+). Furthermore, to avoid the visual errors caused by naked eye observation, a previously developed volumetric bar-chart chip (V-Chip) was used to quantitatively detect UO2(2+) concentrations in water by encapsulating Au-Pt nanoparticles in the hydrogel. The UO2(2+) concentrations were visually quantified from the travelling distance of ink-bar on the V-Chip. The method can be used for portable and quantitative detection of uranium in field applications without skilled operators and sophisticated instruments.

Enzyme-Encapsulated Liposome-Linked Immunosorbent Assay Enabling Sensitive Personal Glucose Meter Readout for Portable Detection of Disease Biomarkers.

There is considerable demand for sensitive, selective, and portable detection of disease-associated proteins, particularly in clinical practice and diagnostic applications. Portable devices are highly desired for detection of disease biomarkers in daily life due to the advantages of being simple, rapid, user-friendly, and low-cost. Herein we report an enzyme-encapsulated liposome-linked immunosorbent assay for sensitive detection of proteins using personal glucose meters (PGM) for portable quantitative readout. Liposomes encapsulating a large amount of amyloglucosidase or invertase are surface-coated with recognition elements such as aptamers or antibodies for target recognition. By translating molecular recognition signal into a large amount of glucose with the encapsulated enzyme, disease biomarkers such as thrombin or C-reactive protein (CRP) can be quantitatively detected by a PGM with a high detection limit of 1.8 or 0.30 nM, respectively. With the advantages of portability, ease of use, and low-cost, the method reported here has potential for portable and quantitative detection of various targets for different POC testing scenarios, such as rapid diagnosis in clinic offices, health monitoring at the bedside, and chemical/biochemical safety control in the field.