A fast and ultrasensitive ELISA based on rolling circle amplification.

A highly sensitive ELISA is critical for early diagnosis and biomarker discovery of various diseases. Although various ELISA technologies have been developed with high sensitivity, they are limited by poor repeatability, high cost, the dependence on complex equipment and/or a prolonged reaction time. To this end, we developed a fast and ultrasensitive ELISA (termed RELISA) based on rolling circle amplification (RCA) and enzymatic signal amplification. The RELISA is established on the traditional ELISA, with only one more RCA step that can be accomplished within 10 minutes. The prolonged single strand DNA (ssDNA) from RCA is able to enrich abundant horseradish peroxidase conjugate (HRP) modified detection probes. Consequently, the intensive HRP is able to catalyze TMB-H2O2 to produce significantly enhanced colorimetric signals. With CEACAM-7 as a model biomarker, the RELISA achieves the limit of detection as low as 2.82 pg mL-1, which is ∼50 times higher than that of the traditional ELISA. Therefore, we envision that the developed RELISA would be a powerful tool for the early diagnosis of various major diseases.

A digitalized isothermal nucleic acid testing platform based on a pump-free open droplet array microfluidic chip.

Digital PCR has shown great potential for quantitative nucleic acid testing (NAT), but most existing platforms are dependent on large auxiliary equipment (e.g., vacuum pump, amplification instrument, fluorescence microscope) to achieve target dispersion, amplification, signal capture and result analysis. Such complex, expensive and bulky NAT platforms have limited their applications in resource-limited areas, especially for point-of-care testing (POCT). In this work, we designed a digital isothermal NAT platform based on a pump-free open droplet array microfluidic chip. A pump-free microfluidic chip was developed based on an open microdroplet array in the form of thousands of independent microdroplets for spontaneous sample dispersion, without the need for external power. Combined with a handheld fluorescent signal reader based on a smartphone, this digital NAT platform can accurately quantify as low as 1 copy per µL of λDNA. Therefore, our integrated NAT platform, as a potable, robust and low-cost tool for highly accurate NA quantitative analysis, holds great potential for POCT applications.

An upconversion nanoparticle-based photostable FRET system for long-chain DNA sequence detection.

The fluorescence resonance energy transfer (FRET)-based diagnosis method has been widely used in fast and accurate diagnosis. However, the traditional FRET-based diagnosis method is unable to detect long-chain DNA sequences, due to the limitation of the distance between the donor and acceptor, while the long-chain DNA sequence enables higher selectivity and is quite important for confirming many major diseases. Therefore, it is urgently needed to develop an efficient FRET system for long-chain DNA detection. Herein a 'head-to-tail' structure was developed using NaYF4:Yb,Er nanoparticles as the energy donor and gold nanoparticles (AuNPs) as the acceptor to detect long-chain oligonucleotides sequences (i.e., HIV DNA, 52 bp). We modified NaYF4:Yb,Er nanoparticles with carboxylic acid groups by using poly(acrylic acid) to enhance its hydrophilic and then covalently attached 5 'end of capture oligonucleotides strand to the surface of the particles. In the presence of target HIV DNA, gold nanoparticles with reported DNA were brought close to NaYF4:Yb,Er nanoparticles upon 'head-to-tail' sandwich hybridization with target HIV DNA, resulting in an efficient FRET. Moreover, benefited from both photostable nanoparticles of UCNPs and AuNPs, the photobleaching issue has also been settled down. This developed method possesses high selectivity, high sensitivity, and reached a nanomolar limitation level. To our knowledge, it is the first time to report a 'head-to-tail' structure FRET system for detecting long-chain DNA sequences.

Capillary blood for point-of-care testing.

Clinically, blood sample analysis has been widely used for health monitoring. In hospitals, arterial and venous blood are utilized to detect various disease biomarkers. However, collection methods are invasive, painful, may result in injury and contamination, and skilled workers are required, making these methods unsuitable for use in a resource-limited setting. In contrast, capillary blood is easily collected by a minimally invasive procedure and has excellent potential for use in point-of-care (POC) health monitoring. In this review, we first discuss the differences among arterial blood, venous blood, and capillary blood in terms of the puncture sites, components, sample volume, collection methods, and application areas. Additionally, we review the most recent advances in capillary blood-based commercial products and microfluidic instruments for various applications. We also compare the accuracy of microfluidic-based testing with that of laboratory-based testing for capillary blood-based disease diagnosis at the POC. Finally, we discuss the challenges and future perspectives for developing capillary blood-based POC instruments.

Antiproliferative Activity and Cellular Uptake of Evodiamine and Rutaecarpine Based on 3D Tumor Models.

Evodiamine (EVO) and rutaecarpine (RUT) are promising anti-tumor drug candidates. The evaluation of the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids of cancer cells would better recapitulate the native situation and thus better reflect an in vivo response to the treatment. Herein, we employed the 3D culture of MCF-7 and SMMC-7721 cells based on hanging drop method and evaluated the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids, and compared the results with those obtained from 2D monolayers. The drugs' IC50 values were significantly increased from the range of 6.4-44.1 µM in 2D monolayers to 21.8-138.0 µM in 3D multicellular spheroids, which may be due to enhanced mass barrier and reduced drug penetration in 3D models. The fluorescence of EVO and RUT was measured via fluorescence spectroscopy and the cellular uptake of both drugs was characterized in 2D tumor models. The results showed that the cellular uptake concentrations of RUT increased with increasing drug concentrations. However, the EVO concentrations uptaken by the cells showed only a small change with increasing drug concentrations, which may be due to the different solubility of EVO and Rut in solvents. Overall, this study provided a new vision of the anti-tumor activity of EVO and RUT via 3D multicellular spheroids and cellular uptake through the fluorescence of compounds.

DNAzyme-based ultrasensitive immunoassay: Recent advances and emerging trends.

Immunoassay, as the most commonly used method for protein detection, is simple to operate and highly specific. Sensitivity improvement is always the thrust of immunoassays, especially for the detection of trace quantities. The emergence of artificial enzyme, i.e., DNAzyme, provides a novel approach to improve the detection sensitivity of immunoassay. Simultaneously, its advantages of simple synthesis and high stability enable low cost, broad applicability and long shelf life for immunoassay. In this review, we summarized the recent advances in DNAzyme-based immunoassay. First, we summarized the existing different DNAzymes based on their catalytic activities. Next, the common signal amplification strategies used for DNAzyme-based immunoassays were reviewed to cater to diverse detection requirements. Following, the wide applications in disease diagnosis, environmental monitoring and food safety were discussed. Finally, the current challenges and perspectives on the future development of DNAzyme-based immunoassays were also provided.

Polydopamine Nanoparticles-Based Three-Line Lateral Flow Immunoassay for COVID-19 Detection.

Currently, the global trend of several hundred thousand new confirmed COVID-19 patients per day has not abated significantly. Serological antibody detection has become an important tool for the self-screening of people. While the most commonly used colorimetric lateral flow immunoassay (LFIA) methods for the detection of COVID-19 antibodies are limited by low sensitivity and a lack of quantification ability. This leads to poor accuracy in the screening of early COVID-19 patients. Therefore, it is necessary to develop an accurate and sensitive autonomous antibody detection technique that will effectively reduce the COVID-19 infection rate. Here, we developed a three-line LFIA immunoassay based on polydopamine (PDA) nanoparticles for COVID-19 IgG and IgM antibodies detection to determine the degree of infection. The PDA-based three-line LFIA has a detection limit of 1.51 and 2.34 ng/mL for IgM and IgG, respectively. This assay reveals a good linearity for both IgM and IgG antibodies detection and is also able to achieve quantitative detection by measuring the optical density of test lines. In comparison, the commercial AuNP-based LFIA showed worse quantification results than the developed PDA-based LFIA for low-concentration COVID-19 antibody samples, making it difficult to distinguish between negative and positive samples. Therefore, the developed PDA-based three-line LFIA platform has the accurate quantitative capability and high sensitivity, which could be a powerful tool for the large-scale self-screening of people.

Similar color analysis based on deep learning (SCAD) for multiplex digital PCR via a single fluorescent channel.

Digital PCR (dPCR) has recently attracted great interest due to its high sensitivity and accuracy. However, the existing dPCR depends on multicolor fluorescent dyes and multiple fluorescent channels to achieve multiplex detection, resulting in increased detection cost and limited detection throughput. Here, we developed a deep learning-based similar color analysis method, namely SCAD, to achieve multiplex dPCR in a single fluorescent channel. As a demonstration, we designed a microwell chip-based diplex dPCR system for detecting two genes (blaNDM and blaVIM) with two kinds of green fluorescent probes, whose emission colors are difficult to discriminate by traditional fluorescence intensity-based methods. To verify the possibility of deep learning algorithms to distinguish the similar colors, we first applied t-distributed stochastic neighbor embedding (tSNE) to make a clustering map for the microwells with similar fluorescence. Then, we trained a Vision Transformer (ViT) model on 10 000 microwells with two similar colors and tested it with 262 202 microwells. Lastly, the trained model was proven to have highly accurate classification ability (>98% for both the training set and the test set) and precise quantification ability on both blaNDM and blaVIM (ratio difference <0.10). We envision that the developed SCAD method would significantly expand the detection throughput of dPCR without the need for other auxiliary equipment.

Artificial intelligence-assisted colorimetric lateral flow immunoassay for sensitive and quantitative detection of COVID-19 neutralizing antibody.

Currently, vaccination is the most effective medical measure to improve group immunity and prevent the rapid spread of COVID-19. Since the individual difference of vaccine effectiveness is inevitable, it is necessary to evaluate the vaccine effectiveness of every vaccinated person to ensure the appearance of herd immunity. Here, we developed an artificial intelligent (AI)-assisted colorimetric polydopamine nanoparticle (PDA)-based lateral flow immunoassay (LFIA) platform for the sensitive and accurate quantification of neutralizing antibodies produced from vaccinations. The platform integrates PDA-based LFIA and a smartphone-based reader to test the neutralizing antibodies in serum, where an AI algorithm is also developed to accurately and quantitatively analyze the results. The developed platform achieved a quantitative detection with 160 ng/mL of detection limit and 625-10000 ng/mL of detection range. Moreover, it also successfully detected totally 50 clinical serum samples, revealing a great consistency with the commercial ELISA kit. Comparing with commercial gold nanoparticle-based LFIA, our PDA-based LFIA platform showed more accurate quantification ability for the clinical serum. Therefore, we envision that the AI-assisted PDA-based LFIA platform with sensitive and accurate quantification ability is of great significance for large-scale evaluation of vaccine effectiveness and other point-of-care immunoassays.

Fully integrated microfluidic devices for qualitative, quantitative and digital nucleic acids testing at point of care.

Benefiting from emerging miniaturized and equipment-free nucleic acid testing (NAT) technologies, fully integrated NAT devices at point of care (POC) with the capability of "sample-in-answer-out" are proceeding at a break-neck speed to eliminate complex operations and reduce the risk of contamination. Like the development of polymerase chain reaction (PCR) technology (the standard technique for NAT), the detection signal of fully integrated NAT devices has evolved from qualitative to quantitative and recently to digital readout, aiming at expanding their extensive applications through gradually improving detection sensitivity and accuracy. This review firstly introduces the existing commercial products, and then illustrates recent fully integrated microfluidic devices for NAT at POC from the aspect of detection signals (i.e., qualitative, quantitative and digital). Importantly, the key issues of existing commercial products and the main challenges between scientific research and product development are discussed. On this basis, we envision that the MARCHED (miniaturized, automatic, reagent-preloaded, commercializable, high-throughput, environment-independent and disposable) NAT devices are expected to be realized in the near future.

Taqman-MGB nanoPCR for Highly Specific Detection of Single-Base Mutations.

PURPOSE: Detection of single-base mutations is important for real-time monitoring of tumor progression, therapeutic effects, and drug resistance. However, the specific detection of single-base mutations from excessive wild-type background sequences with routine PCR technology remains challenging. Our objective is to develop a simple and highly specific qPCR-based single-base mutation detection method. METHODS: Using EGRF T790M as a model, gold nanoparticles at different concentrations were separately added into the Taqman-MGB qPCR system to test specificity improvement, leading to the development of the optimal Taqman-MGB nanoPCR system. Then, these optimal conditions were used to test the range of improvement in the specificity of mutant-type and wild-type templates and the detection limit of mutation abundances in a spiked sample. RESULTS: The Taqman-MGB nanoPCR was established based on the traditional qPCR, with significantly suppressed background noise and improved specificity for single-base mutation detection. With EGFR T790M as a template, we demonstrated that our Taqman-MGB nanoPCR system could improve specificity across a wide concentration range from 10-9 µM to 10 µM and detect as low as 0.95% mutation abundance in spiked samples, which is lower than what the traditional Taqman-MGB qPCR and existing PCR methods can detect. Moreover, we also proposed an experimentally validated barrier hypothesis for the mechanism of improved specificity. CONCLUSION: The developed Taqman-MGB nanoPCR system could be a powerful tool for clinical single-base mutation detection.

A Portable Digital Loop-Mediated Isothermal Amplification Platform Based on Microgel Array and Hand-Held Reader.

Digital polymerase chain reaction (dPCR) has found widespread applications in molecular diagnosis of various diseases owing to its sensitive single-molecule detection capability. However, the existing dPCR platforms rely on the auxiliary procedure to disperse DNA samples, which needs complicated operation, expensive apparatus, and consumables. Besides, the complex and costly dPCR readers also impede the applications of dPCR for point-of-care testing (POCT). Herein, we developed a portable digital loop-mediated isothermal amplification (dLAMP) platform, integrating a microscale hydrogel (microgel) array chip for sample partition, a miniaturized heater for DNA amplification, and a hand-held reader for digital readout. In the platform, the chip with thousands of isolated microgels holds the capability of self-absorption and partition of DNA samples, thus avoiding auxiliary equipment and professional personnel operations. Using the integrated dLAMP platform, λDNA templates have been quantified with a good linear detection range of 2-1000 copies/µL and a detection limit of 1 copy/µL. As a demonstration, the epidermal growth factor receptor L858R gene mutation, a crucial factor for the susceptibility of the tyrosine kinase inhibitor in non-small-cell lung cancer treatment, has been accurately identified by the dLAMP platform with a spiked plasma sample. This work shows that the developed dLAMP platform provides a low-cost, facile, and user-friendly solution for the absolute quantification of DNA, showing great potential for the POCT of nucleic acids.

Quantifying and Adjusting Plasmon-Driven Nano-Localized Temperature Field around Gold Nanorods for Nucleic Acids Amplification.

Fast nucleic acid (NA) amplification has found widespread biomedical applications, where high thermocycling rate is the key. The plasmon-driven nano-localized thermocycling around the gold nanorods (AuNRs) is a promising alternative, as the significantly reduced reaction volume enables a rapid temperature response. However, quantifying and adjusting the nano-localized temperature field remains challenging for now. Herein, a simple method is developed to quantify and adjust the nano-localized temperature field around AuNRs by combining experimental measurement and numerical simulation. An indirect method to measure the surface temperature of AuNRs is first developed by utilizing the temperature-dependent stability of Authiol bond. Meanwhile, the relationship of AuNRs' surface temperature with the AuNRs concentration and laser intensity, is also studied. In combination with thermal diffusion simulation, the nano-localized temperature field under the laser irradiation is obtained. The results show that the restricted reaction volume (≈aL level) enables ultrafast thermocycling rate (>104  °C s-1 ). At last, a duplex-specific nuclease (DSN)-mediated isothermal amplification is successfully demonstrated within the nano-localized temperature field. It is envisioned that the developed method for quantifying and adjusting the nano-localized temperature field around AuNRs is adaptive for various noble metal nanostructures and will facilitate the development of the biochemical reaction in the nano-localized environment.

Microneedle patch for the ultrasensitive quantification of protein biomarkers in interstitial fluid.

The detection and quantification of protein biomarkers in interstitial fluid is hampered by challenges in its sampling and analysis. Here we report the use of a microneedle patch for fast in vivo sampling and on-needle quantification of target protein biomarkers in interstitial fluid. We used plasmonic fluor-an ultrabright fluorescent label-to improve the limit of detection of various interstitial fluid protein biomarkers by nearly 800-fold compared with conventional fluorophores, and a magnetic backing layer to implement conventional immunoassay procedures on the patch and thus improve measurement consistency. We used the microneedle patch in mice for minimally invasive evaluation of the efficiency of a cocaine vaccine, for longitudinal monitoring of the levels of inflammatory biomarkers, and for efficient sampling of the calvarial periosteum-a challenging site for biomarker detection-and the quantification of its levels of the matricellular protein periostin, which cannot be accurately inferred from blood or other systemic biofluids. Microneedle patches for the minimally invasive collection and analysis of biomarkers in interstitial fluid might facilitate point-of-care diagnostics and longitudinal monitoring.

Ultrafast Photonic PCR Based on Photothermal Nanomaterials.

Over the past few decades, PCR has been the gold standard for detecting nucleic acids (NAs) in various biomedical fields. However, there are several limitations associated with conventional PCR, such as complicated operation, need for bulky equipment, and, in particular, long thermocycling time. Emerging nanomaterials with photothermal effects have shown great potential for developing a new generation of PCR: ultrafast photonic PCR. Here, we review recent applications of photothermal nanomaterials in ultrafast photonic PCR. First, we introduce emerging photothermal nanomaterials and their light-to-heat energy conversion process in photonic PCR. We then review different photothermal nanomaterial-based photonic PCRs and compare their merits and drawbacks. Finally, we summarize existing challenges with photonic PCR and hypothesize its promising future research directions.

Paper-based point-of-care immunoassays: Recent advances and emerging trends.

Immunoassay has been routinely used in hospitals and central labs. Nowadays, to further meet the requirement of widespread applications of immunoassays, it is urgently needed to produce a simplified, rapid and low-cost immunoassay to perform tests on site. To this end, paper-based point-of-care (POC) immunoassays have attracted intensive interests in recent years. In this paper, we present a comprehensive review of the recent advances and emerging trends of paper-based POC immunoassays, including the fundamental components and work principles, various detection mechanisms and applications, and existing commercialized devices/products. At last, we envision three promising development directions for paper-based POC immunoassays.

A portable and universal upconversion nanoparticle-based lateral flow assay platform for point-of-care testing.

Upconversion nanoparticle-based lateral flow assays (UCNP-LFAs) have attracted significant attention in point-of-care testing (POCT) applications, due to the long-term photostability and enhanced signal-to-background noise ratio. The existing UCNP-LFAs generally require peripheral equipment for exciting fluorescent signals and reading out fluorescence results, which are generally bulky and expensive. Herein, we developed a miniaturized and portable UCNP-LFA platform, which is composed of a LFA detection system, an UCNP-LFA reader and a smartphone-assisted UCNP-LFA analyzer. The LFA detection system is based on three types of UCNPs for multiplexed detection. The reader has a dimension of 24.0 cm × 9.4 cm × 5.4 cm (L × W × H) and weight of 0.9 kg. The analyzer based on the custom-designed software of a smartphone (termed as UCNP-LFA analyzer) can get the quantitative analysis results in a real-time manner. We demonstrated the universality of this platform by highly sensitive and quantitative detections of several kinds of targets, including small molecule (ochratoxin A, OTA), heavy metal ion (Hg2+), bacteria (salmonella, SE), nucleic acid (hepatitis B virus, HBV) and protein (growth stimulation expressed gene 2, ST-2). Our developed UCNP-LFA platform holds great promise for applications in disease diagnostics, environmental pollution monitoring and food safety at the point of care.

Point-of-Care Periodontitis Testing: Biomarkers, Current Technologies, and Perspectives.

Periodontitis has become one of the most universal chronic inflammatory diseases worldwide. Subclinical symptom progression, ultimately leading to permanent damage, calls for early diagnosis and long-term monitoring. However, traditional clinical diagnostic methods are complex and expensive, and cannot meet these requirements. Recently, with more biomarkers and the development of new technologies, various point-of-care testing (POCT) platforms have been developed for periodontitis diagnosis and monitoring. These are easy to perform, rapid, low-cost, and are perfectly suited for high-frequency diagnosis of periodontitis at the point-of-care (POC). We summarize existing biomarkers of different periodontitis stages and recent developed POCT platforms (including lab-on-a-chip, paper-based platforms, and chairside tests), discuss their existing challenges and future potential, and provide some inspiration and guidelines for future POC periodontitis testing.

Improved LFIAs for highly sensitive detection of BNP at point-of-care.

Heart failure (HF) has become a major cause of morbidity and mortality with a significant global economic burden. Although well-established clinical tests could provide early diagnosis, access to these tests is limited in developing countries, where a relatively higher incidence of HF is present. This has prompted an urgent need for developing a cost-effective, rapid and robust diagnostic tool for point-of-care (POC) detection of HF. Lateral flow immunoassay (LFIA) has found widespread applications in POC diagnostics. However, the low sensitivity of LFIA limits its ability to detect important HF biomarkers (e.g., brain natriuretic peptide [BNP]) that are normally present in low concentration in blood. To address this issue, we developed an improved LFIA by optimizing the gold nanoparticle (GNP)-antibody conjugate conditions (e.g., the conjugate pH and the amount of added antibody), the diameter of GNP and the concentration of antibody embedded on the test line and modifying the structure of test strip. Through these improvements, the proposed test strip enabled the detection of BNP down to 0.1 ng/mL within 10-15 min, presenting ~15-fold sensitivity enhancement over conventional lateral flow assay. We also successfully applied our LFIA in the analysis of BNP in human serum samples, highlighting its potential use for clinical assessment of HF. The developed LFIA for BNP could rapidly rule out HF with the naked eye, offering tremendous potential for POC test and personalized medicine.