In situ Raman enhancement strategy for highly sensitive and quantitative lateral flow assay.

As a paper-based analytical platform, lateral flow assay (LFA) gets benefit from the rapid analysis, low cost, high selectivity, good stability, and user-friendliness, and thus has been widely used in rapid screening or assisted diagnosis. Nevertheless, LFA still suffers from low detection sensitivity via the naked eye, limiting its applications to qualitative and semi-quantitative tests. To enhance the signal readout, various nanoparticle signal tags have been employed to replace traditional colloidal gold nanoparticles (AuNPs), such as fluorescent nanoparticles (FNPs), magnetic nanoparticles (MNPs), and Raman reporter-labeled nanoparticles. In particular, Raman reporter-labeled nanoparticles are extremely sensitive due to remarkable signal enhancement effect on metal surface. However, the application of LFA is still hampered by the poor stability of Raman reporter-labeled nanoparticles. Herein, we developed an in situ Raman enhancement strategy to create a surface-enhanced Raman scattering (SERS) signal on the AuNPs, shortened as "i-SERS," which not only preserves the original advantages of the colloidal gold strip (AuNPs-LFA), but also realizes highly sensitive and quantitative detection. We applied the i-SERS for procalcitonin (PCT) detection. The experimental process takes only 16 min, and the limit of detection (LOD) is 0.03 ng mL-1, far below the value using AuNPs-LFA. These results indicate that i-SERS assay was highly sensitive and suitable for the rapid detection of PCT.

A smartphone-assisted pressure-measuring-based diagnosis system for acute myocardial infarction diagnosis.

BACKGROUND: Acute myocardial infarction (AMI), usually caused by atherosclerosis of coronary artery, is the most severe manifestation of coronary artery disease which results in a large amount of death annually. A new diagnosis approach with high accuracy, reliability and low measuring-time-consuming is essential for AMI quick diagnosis. PURPOSE: The objective of this study was to develop a new point-of-care testing system with high accuracy and reliability for AMI quick diagnosis. PATIENTS AND METHODS: 50 plasma samples of acute myocardial infarction patients were analyzed by developed Smartphone-Assisted Pressure-Measuring-Based Diagnosis System (SPDS). The concentration of substrate was firstly optimized. The effect of antibody labeling and matrix solution on measuring result were then evaluated. And standard curves for cTnI, CK-MB and Myo were built for clinical sample analysis. The measuring results of 50 clinical samples were finally evaluated by comparing with the measuring result obtained by CLIA. RESULTS: The concentration of substrate H2O2 was firstly optimized as 30% to increase measuring signal. A commercial serum matrix was chosen as the matrix solution to dilute biomarkers for standard curve building to minimize matrix effect on the accuracy of clinical plasma sample measuring. The standard curves for cTnI, CK-MB and Myo were built, with measuring dynamic range of 0-25 ng/mL, 0-33 ng/mL and 0-250 ng/mL, and limit of detection of 0.014 ng/mL, 0.16 ng/mL and 0.85 ng/mL respectively. The measuring results obtained by the developed system of 50 clinical plasma samples for three biomarkers matched well with the results obtained by chemiluminescent immunoassay. CONCLUSION: Due to its small device size, high sensitivity and accuracy, SPDS showed a bright potential for point-of-care testing (POCT) applications.

Staining Traditional Colloidal Gold Test Strips with Pt Nanoshell Enables Quantitative Point-of-Care Testing with Simple and Portable Pressure Meter Readout.

Traditional immunochromatographic test strips based on colloidal gold are effective devices for portable and low-cost point-of-care (POC) testing. Nevertheless, they still suffer from the limitation of qualitative or semiquantitative tests via naked-eye detection. Replacement of gold with other signal entities, such as magnetic particles or fluorescent particles, requires professional instrumentation to obtain quantitative results. A pressure-based assay with platinum nanoparticles (PtNPs) can provide quantitative results using a portable pressure meter but is also hampered by the long-term instability of PtNPs. Consequently, we developed a Pt-staining method based on test strips to create platinum nanoshells on the surface of colloidal gold. This method not only preserves the original advantages of colloidal gold with easy synthesis and decoration but also introduces PtNPs with excellent catalytic activity as signal labels to achieve sensitive quantitative detection. Myoglobin was tested as a model target, and the limit of detection was 5.47 ng/mL in 20% diluted serum samples, which satisfies the requirements for clinical monitoring of acute myocardial infarction. In addition, the two most common colloidal gold strips available in the marketplace were applied to demonstrate the compatibility of Pt-staining. Taking advantage of low cost, user-friendliness, compatibility, simplicity, and stability, colloidal gold test strips with Pt-staining are expected to satisfy the need for quantitative POC testing of biomarkers, especially in resource-limited regions.

Lateral flow assay with pressure meter readout for rapid point-of-care detection of disease-associated protein.

Paper-based assays such as lateral flow assays are good candidates for portable diagnostics owing to their user-friendly format and low cost. In terms of analytical detection, lateral flow assays usually require dedicated instruments to obtain quantitative results. Here we demonstrate a lateral flow assay with handheld pressure meter readout for the rapid detection of disease-related protein with high sensitivity and selectivity. Based on the pressure change produced by the catalytic reaction of Pt nanoparticles related to the concentration of the target, a quantitative reaction platform was established. During the lateral flow assay, the Pt nanoparticles are aggregated in the test line to form a gray band by biomolecular recognition and finally convert the recognition signal into highly sensitive pressure readout for quantitative analysis. Without sophisticated instrumentation and complicated operations, the whole detection process can be completed within 20 minutes. The limit of detection for myoglobin (2.9 ng mL-1 in diluted serum samples) meets the requirements of clinical monitoring. With the advantages of low cost, ease of operation, high sensitivity and selectivity, the method represents a versatile platform for point-of-care testing of disease biomarkers.

Microwell Array Method for Rapid Generation of Uniform Agarose Droplets and Beads for Single Molecule Analysis.

Compartmentalization of aqueous samples in uniform emulsion droplets has proven to be a useful tool for many chemical, biological, and biomedical applications. Herein, we introduce an array-based emulsification method for rapid and easy generation of monodisperse agarose-in-oil droplets in a PDMS microwell array. The microwells are filled with agarose solution, and subsequent addition of hot oil results in immediate formation of agarose droplets due to the surface-tension of the liquid solution. Because droplet size is determined solely by the array unit dimensions, uniform droplets with preselectable diameters ranging from 20 to 100 µm can be produced with relative standard deviations less than 3.5%. The array-based droplet generation method was used to perform digital PCR for absolute DNA quantitation. The array-based droplet isolation and sol-gel switching property of agarose enable formation of stable beads by chilling the droplet array at -20 °C, thus, maintaining the monoclonality of each droplet and facilitating the selective retrieval of desired droplets. The monoclonality of droplets was demonstrated by DNA sequencing and FACS analysis, suggesting the robustness and flexibility of the approach for single molecule amplification and analysis. We believe our approach will lead to new possibilities for a great variety of applications, such as single-cell gene expression studies, aptamer selection, and oligonucleotide analysis.

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.

A Shake&Read distance-based microfluidic chip as a portable quantitative readout device for highly sensitive point-of-care testing.

Despite the rapid development of point-of-care testing (POCT) devices in recent years, quantitative POCT is still not readily available. Herein, we developed a simple, disposable and equipment-free quantitative POCT platform, the Shake&Read distance-based microfluidic Chip (S&R-µChip), for visual quantitative POCT and demonstrated its use in Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of disease biomarkers.

Translating Molecular Recognition into a Pressure Signal to enable Rapid, Sensitive, and Portable Biomedical Analysis.

Herein, we demonstrate that a very familiar, yet underutilized, physical parameter­gas pressure­can serve as signal readout for highly sensitive bioanalysis. Integration of a catalyzed gas-generation reaction with a molecular recognition component leads to significant pressure changes, which can be measured with high sensitivity using a low-cost and portable pressure meter. This new signaling strategy opens up a new way for simple, portable, yet highly sensitive biomedical analysis in a variety of settings.

Microfluidic approaches to rapid and efficient aptamer selection.

With their advantages as molecular recognition elements, aptamers have been extensively studied and used for bioanalytical and biomedical applications. However, the process of enrichment and screening of aptamers remains a bottleneck for aptamer development. Recently, microfluidic methods have been increasingly used for rapid and efficient aptamer selection, showing their remarkable advantages over conventional methods. This review briefly introduces aptamers and their advantages. The conventional process of generating aptamers is discussed, followed by the analysis of the key obstacles to efficient aptamer selection. Microfluidic methods for highly efficient enrichment and screening of aptamers are reviewed in detail.

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.

A cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing.

Accurate sensing of the extracellular pH is a very important yet challenging task in biological and clinical applications. This paper describes the development of an amphiphilic lipid-DNA molecule as a simple yet useful cell-surface-anchored ratiometric fluorescent probe for extracellular pH sensing. The lipid-DNA probe, which consists of a hydrophobic diacyllipid tail and a hydrophilic DNA strand, is modified with two fluorescent dyes; one is pH-sensitive as pH indicator and the other is pH-insensitive as an internal reference. The lipid-DNA probe showed sensitive and reversible response to pH change in the range of 6.0-8.0, which is suitable for most extracellular studies. In addition, based on simple hydrophobic interactions with the cell membrane, the lipid-DNA probe can be easily anchored on the cell surface with negligible cytotoxicity, excellent stability, and unique ratiometric readout, thus ensuring its accurate sensing of extracellular pH. Finally, this lipid-DNA-based ratiometric pH indicator was successfully used for extracellular pH sensing of cells in 3D culture environment, demonstrating the potential applications of the sensor in biological and medical studies.

A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection.

Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single ß-galatosidase (ß-Gal) molecules and the fluorogenic substrate fluorescein di-ß-D-galactopyranoside were injected from two separated inlets to form uniform 20 µm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of ß-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the ß-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of ß-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in uniform picoliter droplets paves the way to microdroplet based digital detection of proteins.

Facile and rapid generation of large-scale microcollagen gel array for long-term single-cell 3D culture and cell proliferation heterogeneity analysis.

Microfabricated devices are suitable for single-cell analysis due to their high throughput, compatible dimensions and controllable microenvironment. However, existing devices for single-cell culture and analysis encounter some limitations, such as nutrient depletion, random cell migration and complicated fluid shear influence. Moreover, most of the single-cell culture and analysis devices are based on 2D cell culture conditions, even though 3D cell culture methods have been demonstrated to better mimic the real cell microenvironment in vivo. To solve these problems, herein we develop a microcollagen gel array (µCGA) based approach for high-throughput long-term single-cell culture and single-cell analysis under 3D culture conditions. Type-I collagen, a well-established 3D cell culture medium, was used as the scaffold for 3D cell growth. A 2 × 2 cm PDMS chip with 10 000 µCGA units was fabricated to encapsulate thousands of single cells in less than 15 min. Single cells were able to be confined and survive in µCGA units for more than 1 month. The capability of large-scale and long-term single-cell 3D culture under open culture conditions allows us to study cellular proliferation heterogeneity and drug cytotoxicity at the single-cell level. Compared with existing devices for single-cell analysis, µCGA solves the problems of nutrient depletion and random cellular migration, avoids the influence of complicated fluid shear, and mimics the real 3D growth environment in vivo, thereby providing a feasible 3D long-term single-cell culture method for single-cell analysis and drug screening.

A T7 exonuclease-assisted cyclic enzymatic amplification method coupled with rolling circle amplification: a dual-amplification strategy for sensitive and selective microRNA detection.

A T7 exonuclease-assisted cyclic enzymatic amplification method (CEAM) was combined with rolling circle amplification (RCA) to develop a RCA-CEAM dual amplification method for ultrasensitive detection of microRNA with excellent selectivity.

Cyclic enzymatic amplification method (CEAM) based on exonuclease III for highly sensitive bioanalysis.

Nucleic acid molecular probes (NAMPs) have been widely used in the sensing of various chemical and biological substances, as well as physical parameters. However, for traditional nucleic acid molecular probes, the stoichiometric 1:1 binding ratio limits the signal enhancement and thus the sensitivity of the assay. In order to overcome this problem, the cyclic enzymatic amplification method (CEAM) based on exonuclease III has been applied in optical and electrical detection of DNA, proteins and small molecules with excellent sensitivity, selectivity, versatility and simplicity. In this review, the working principle of CEAM is first introduced, followed by the applications of CEAM using different output signals for various analytes. Finally, experimental designs and procedures of CEAM are discussed in detail using displacing probes-based CEAM and linear molecular beacons (LMBs)-based CEAM as two examples.

Graphene oxide protected nucleic acid probes for bioanalysis and biomedicine.

Recently, the binding ability of DNA on GO and resulting nuclease resistance have attracted increasing attention, leading to new applications both in vivo and in vitro. In vivo, nucleic acids absorbed on GO can be effectively protected from enzymatic degradation and biological interference in complicated samples, making it useful for targeted delivery, gene regulation, intracellular detection and imaging with high uptake efficiencies, high intracellular stability, and very low toxicity. In vitro, the adsorption of ssDNA on GO surface and desorption of dsDNA or well-folded ssDNA from GO surface result in the protection and deprotection of DNA from nucleic digestion, respectively, which has led to target-triggered cyclic enzymatic amplification methods (CEAM) for amplified detection of analytes with sensitivity 2-3 orders of magnitude higher than that of 1:1 binding strategies. This Concept article explores some of the latest developments in this field.

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Single-molecule emulsion PCR in microfluidic droplets.

The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.