Fluorescent Lateral Flow Research Articles

Development and clinical evaluation of a quantitative fluorescent immunoassay for detecting canine CRP

ABSTRACT Canine C-reactive protein (cCRP) is one of the major positive acute phase proteins in dogs and is commonly measured to detect and monitor systemic inflammation as well as the efficacy of treatment. Traditional methods for testing cCPR, including enzyme-linked immunosorbent assay (ELISA), have some drawbacks, such as a long time for diagnosis and the requirement of well-equipped laboratories. Therefore, there is a need for a rapid and precise diagnostic test for cCRP at point-of-care. This study assessed the accuracy, precision, and validated clinical effectiveness of a diagnostic test based on fluorescent lateral flow immunoassay to detect cCRP. For the standard cCRP concentration ranging from 0 to 200 μg/mL, the cCRP diagnostic test showed strong linearity with R2 of 0.9977 (p < 0.001), and both inter- and intra-assay CVs were <14%. The limit of detection and limit of quantitation were found to be 4.0 μg/mL and 5.0 μg/mL, respectively. The cCRP serum concentration was evaluated in 21 client-owned dogs and the results were compared to a previously validated ELISA. The Pearson Correlation Coefficient between the diagnostic test kit and ELISA was 0.942 [95% confidence interval: 0.859 to 0.976, p < 0.001], and the Bland–Altman plot indicated a bias of 26.82% [95% limits of agreement: −56.03 to 109.67], indicating a significant correlation and the agreement between the data from the cCRP diagnostic test and ELISA. In conclusion, the fluorescent immunoassay based diagnostic test is a suitable option for rapidly and precisely detecting cCRP in dogs, providing a convenient alternative to traditional methods for diagnosing acute inflammation.

Competitive ratiometric fluorescent lateral flow immunoassay based on dual emission signal for sensitive detection of chlorothalonil

In this study, we develop a competitive ratiometric fluorescent lateral flow immunoassay (CRF-LFIA) based on dual emission fluorescence signal, which has great advantage in visual and quantitative detection of Chlorothalonil (CTN). Red-emitted fluorescent magnetic nanobeads (FMNBs) and green-emitted aggregation-induced emission fluorescent microsphere (AIEFM) are synthesized and conjugated to antibodies and antigens respectively, resulting in competitive binding with the analyte. The ratiometric fluorescence signal which comes from the overlap of these two fluorescence emissions. FMNBs probes also provide immunomagnetic separation (IMS) to enrich the analysts and resist complex matrix effects. This strip generates a visually discernible yellow-to-green fluorescence color change in the presence of CTN (2 ng/mL), which could be incisively observed by naked eye. Moreover, the limit of detection (LOD) reached 0.152 ng/mL by measurement of color (Red-Green-Blue, RGB) signals. Method validation shows a good correlation between CRF-LFIA and LC-MS/MS.

Introduction of multilayered quantum dot nanobeads into competitive lateral flow assays for ultrasensitive and quantitative monitoring of pesticides in complex samples.

A competitive fluorescent lateral flow assay (CFLFA) is proposed for direct, ultrasensitive, quantitative detection of common pesticides imidacloprid (IMI) and carbendazim (CBZ) in complex food samples by using silica-core multilayered quantum dot nanobeads (SiO2-MQB) as liquid fluorescent tags. The SiO2-MQB nanostructure comprises a 200-nm SiO2 core and a shell of hundreds of carboxylated QDs (excitation/emission maxima ~365/631 nm), and can generate better stability, superior dispersibility, and higher luminescence than traditional fluorescent beads, greatly improving the sensitivity of current LFA methods for pesticides. Moreover, using liquid SiO2-MQB directly instead of via the conjugate pad both simplifies the structure of LFA system and improves the efficiency of immunobinding reactions between nanotags and the targets. Applying these methods, the established CFLFA realized the stable and accurate detection of IMI and CBZ in 12 min, with detection limits down to 1.94 and 14.79 pg/mL, respectively. The SiO2-MQB-CFLFA is practicable for application to real food samples (corn, apple, cucumber, and cabbage), and undoubtedly a promising and low-cost tool for on-site monitoring of trace pesticide residues.

A particle-based microfluidic fluorescent lateral flow assay for rapid and sensitive detection of SARS-CoV-2 antibody

Prompt isolation and treatment of patients are effective measures for preventing the spread of infectious diseases. Therefore, a sensitive and early diagnostic test is crucial for controlling pandemics. Herein, we present a tubeless integrated microfluidic lateral flow immunoassay chip for rapid and sensitive detection of SARS-CoV-2 antibodies. Fully automated chip operation is demonstrated by incorporating a dry reagent storage chamber, staggered herringbone micromixer, mobile particle-based detection zone, and spontaneous liquid transportation mechanism. The effects of the position and number of dry reagent patterns on assay time and detection sensitivity were investigated. Under laminar flow conditions, a staggered herringbone mixer was used to achieve rapid and complete mixing of the sample and reagent. In addition, we found that the volumetric flux of the sample and reagent complex over the surface of the particles is a crucial factor for improving detection sensitivity. The limit of detection of an anti-SARS-CoV-2 spike protein monoclonal antibody was determined to be 10.35 ng/mL in assay buffer with a total assay time of 15 min. In the analyte concentration range of 15.625–1000 ng/mL, the linearity and spike-recovery of the assay on serially diluted serum samples were excellent (R2 ≈ 0.99 and recovery rate of 82.9–112 %). The integrated microfluidic immunoassay chip demonstrates rapid and simple operation, which is promising for the diagnosis and monitoring of SARS-CoV-2 antibodies.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

QDs-based fluorescent lateral flow assays for Point-of-care testing of insulin.

Point-of-care testing (POCT) can be performed near the site of the patient to achieve results in a few minutes. Different POCT devices are available in the market, such as microfluidic chips and paper-based lateral flow assays (LFAs). The paper-based LFAs have certain advantages, such as being cheap and disposable, able to detect a wide range of biomolecules, and the fluid flows through them via capillary action eliminating the need for external forces. The LFAs can be optimized for the sensitive and rapid detection of biomolecules. In this study, paper-based fluorescent LFAs platforms using aptamers as the biorecognition molecules were developed for the POCT of insulin. Various parameters were optimized such as concentrations of aptamers, the type of reporter molecules, the volume of sample, and the assay time to quantify insulin levels using a standard LFA reader. The fluorescent LFAs exhibited a linear detection range of 0.1-4 ng.mL-1 with a limit of detection (LOD) 0.1 ng.mL-1. The developed LFAs will help to achieve insulin measurement in a few minutes and will be easy to perform by end-users without the requirement of sophisticated instruments, laboratory set-up, and trained personnel. The developed device will be useful for the measurement of insulin levels in biological samples without the need for pretreatment, reducing the overall cost and time of testing. Moreover, the POCT device were fabricated using paper which is a low-cost (approximately AUD 2 per strip) option and is disposable.Clinical Relevance- POCT monitoring of insulin can facilitate both disease diagnosis and management. The developed LFAs have the capability of rapidly testing insulin concentration within several minutes. It will benefit both patients for at-home daily insulin monitoring and clinicians for hospital rapid insulin testing.

A novel carboxyl polymer-modified upconversion luminescent nanoprobe for detection of prostate-specific antigen in the clinical gray zonebase by flow immunoassay strip.

Prostate specific antigen (PSA) is a widely-used biomarker for the diagnosis, screening, and prognosis of prostate cancer (PCa). It is critical to develop a rapid and convenient method to accurately detect PSA levels, especially when the PSA levels are in the clinical gray area of 4-10ng/mL. We developed a novel upconversion nanoparticle (UCNP)-based fluorescence lateral flow test strip for qualitatively and quantitatively detecting PSA. The carboxyl group-modified UCNPs (UCNP-COOH) were labeled with anti-PSA antibodies via 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as labeling probes to recognize PSA. The fluorescence intensity of the UCNP-probe was then measured with a laser fluorescence scanner. A total of 1397 serum and 20 fingertip blood samples were collected to validate the UCNP strip. A reliable correlation between the area ratio (TC), reflecting the fluorescence intensity of the test/control line, and the PSA concentration was observed (r=0.9986). The dose-dependent luminescence enhancement showed good linearity in the PSA concentration range from 0.1 to 100.0ng/mL with a detection limit of 0.1ng/mL. Our UCNP POCT strip demonstrated excellent accuracy, anti-interference and stability in the gray zone (4-10ng/mL) of PSA clinical application and outperformed other PSA test strips. The UCNP strip showed good consistency with the Roche chemiluminescence assay in 1397 serum samples. It also showed good performance for PSA detection using fingertip blood samples. This novel UCNP-based test strip could be a sensitive and reliable POCT assay to detect PSA, facilitating the diagnosis and surveillance of PCa.

Improving African Swine Fever Surveillance Using Fluorescent Rapid Tests.

African swine fever (ASF) is a viral disease of swine with a huge impact due to its high mortality. Lately, the disease has actively spread around the world, affecting new areas from which it had been eradicated long ago. To date, ASF control is carried out by the implementation of strict biosecurity measures such as the early identification of infected animals. In this work, two fluorescent rapid tests were developed to improve the sensitivity of point-of-care diagnosis of ASF. For antigen (Ag) detection in blood, a double-antibody sandwich fluorescent lateral flow assay (LFA) was developed, employing a newly developed recombinant antibody to the VP72 of the virus. To complement the diagnosis, a double-recognition fluorescent LFA was developed using the VP72 for the detection of specific antibodies (Ab) in sera or blood. Both assays statistically improved the detection of the disease when compared to the commercial colorimetric assays INgezim® ASFV CROM Ag and INgezim® PPA CROM Anticuerpo, respectively, with higher statistical significance between 11 and 39 days post-infection. From the observation of results, it can be concluded that the combination of both Ag-LFA and Ab-LFA assays would facilitate the identification of infected animals, regardless of post-infection time.

Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification with CRISPR/Cpf1 for RNA virus assay

Foot-and-mouth disease virus (FMDV) causes Foot-and-mouth disease, which was listed as a reported disease by Office international des épizooties. It has caused great economic losses to the meat industry. Here, we developed a sensitive, rapid, and visible method based Reverse Transcriptase Enzyme-assisted recombinant isothermal amplification technique (RT-ERA) and CRISPR/Cpf1 for detecting FMDV. RT-ERA achieved simultaneous reverse transcription and amplification in a short time at 37 ℃. Moreover, CRISPR/Cpf1 specifically recognized the products and amplified the signals through the trans-cleavage activity, which converted the DNA signals into fluorescence signals and lateral flow assay strip. Within 30 min, the method detected FMDV as low as 10 copies·μL−1, making it 103 times more sensitive than gel electrophoresis and 10 times more sensitive than reverse transcription quantitative real-time PCR. Moreover, the method had good performance in actual samples. Therefore, this method realized visual detection without the large instruments and showed significant potential for FMDV assay.

A review of advances in aptamer-based cell detection technology.

Since cells are the basic structural and functional units of organisms, the detection or quantitation of cells is one of the most common basic problems in life science research. The established cell detection techniques mainly include fluorescent dye labeling, colorimetric assay, and lateral flow assay, all of which employ antibodies as cell recognition elements. However, the widespread application of the established methods generally dependent on antibodies is limited, because the preparation of antibodies is complicated and time-consuming, and unrecoverable denaturation is prone to occur with antibodies. By contrast, aptamers that are generally selected through the systematic evolution of ligands by exponential enrichment can avoid the disadvantages of antibodies due to their controllable synthesis, thermostability, and long shelf life, etc. Accordingly, aptamers may serve as novel molecular recognition elements like antibodies in combination with various techniques for cell detection. This paper reviews the developed aptamer-based cell detection methods, mainly including aptamer-fluorescent labeling, aptamer-isothermal amplification assay, electrochemical aptamer sensor, aptamer-based lateral flow analysis, and aptamer-colorimetric assay. The principles, advantages, progress of application in cell detection and future development trend of these methods were specially discussed. Overall, different assays are suitable for different detection purposes, and the development of more accurate, economical, efficient, and rapid aptamer-based cell detection methods is always on the road in the future. This review is expected to provide a reference for achieving efficient and accurate detection of cells as well as improving the usefulness of aptamers in the field of analytical applications.

Development of a quantitative fluorescence lateral flow immunoassay (LFIA) prototype for point-of-need detection of anti-Müllerian hormone

ObjectiveAnti-Müllerian Hormone (AMH) is a quantitative marker for ovarian reserve and is used to predict response during ovarian stimulation. Streamlining testing to the clinic or even to the physician's office would reduce inconvenience, turnaround time, patient stress and potentially also the total cost of testing, allowing for more frequent monitoring. In this paper, AMH is used as a model biomarker to describe the rational development and optimization of sensitive, quantitative, clinic-based rapid diagnostic tests. Design and MethodsWe developed a one-step lateral-flow europium (III) chelate-based fluorescent immunoassay (LFIA) for the detection of AMH on a portable fluorescent reader, optimizing the capture/detection antibodies, running buffer, and reporter conjugates. ResultsA panel of commercial calibrators was used to develop a standard curve to determine the analytical sensitivity (LOD = 0.41 ng/ml) and the analytical range (0.41–15.6 ng/ml) of the LFIA. Commercial controls were then tested to perform an initial evaluation of the prototype performance and showed a high degree of precision (Control I CV 2.18%; Control II CV 3.61%) and accuracy (Control I recovery 126%; Control II recovery 103%). Conclusions: This initial evaluation suggests that, in future clinical testing, the AMH LFIA will likely have the capability of distinguishing women with low ovarian reserve (<1 ng/ml AMH) from women with normal (1–4 ng/ml AMH) ovarian reserve. Furthermore, the LFIA demonstrated a wide linear range, indicating the assay's applicability to the detection of other health conditions such as PCOS, which requires AMH measurement at higher concentrations (>6 ng/ml).

Ultrasensitive Hybridization Chain Reaction-Assisted Multisite Exonuclease III Amplification Strategy Combined with a Direct Quantitative Fluorescence Lateral Flow Technique for Multiple Bacterial 16S rRNA Detection.

Accurate and in-time detection of bacteria conduces to preventing their rapid spread around the environment, while a nucleic acid test (NAT) is a powerful tool for early diagnosis of pathogens. Herein, we propose a hybridization chain reaction (HCR)-mediated multisite exonuclease III (Exo-III) amplification strategy (HCR/Exo-III amplifier) to achieve the one-pot and ultrasensitive isothermal amplification of bacterial 16S rRNA and a portable fluorescence detection device (PFD) to directly read signals in a lateral flow assay (LFA). In detail, the target-initiated HCR products present multiple binding sites for triggering the Exo-III amplifier that produces numerous target amplicons. Following that, the target amplicons travel up on the strip and bridge between the DNA-CdTe/CdS probes and the capture DNA to form a positive fluorescence line. After that, the strip is inserted into the PFD to accomplish the fluorescence signal reading. The constructed HCR/Exo-III amplifier-based PFD-LFA implemented the simultaneous and specific detection of three bacteria with a detection limit of a few tenths of fM for synthetic 16S rRNA fragments and dozens of CFU/mL for Staphylococcus aureus, Listeria monocytogenes, and Salmonella typhimurium in pure cultures. The sensing platform features isothermal amplification, convenient operation, and good economy, displaying great potential for on-site testing toward multiple nucleic acid analytes.

Addition of dNTPs can improve the detection sensitivity of catalytic hairpin assembly

Ever since the catalytic hairpin assembly (CHA) circuit has been highlighted as a powerful nucleic acid detection tool, nucleic acid detection methods based on CHA have been widely studied. However, the detection sensitivity of CHA-based methods is insufficient. The relatively high background signals resulting from the spontaneous reaction between the two hairpin probes is one of the major reasons for limiting the sensitivity of CHA. In this study, we established that the addition of deoxynucleotide triphosphates (dNTPs) to the reaction system can significantly reduce the background leakage of CHA. The dNTPs-CHA, coupled with a fluorescence lateral flow assay strip, is used for the rapid and highly sensitive detection of miRNA. It is capable of reliably detecting miRNA in serum samples down to a limit of 100 aM, which is an improvement in the lower detection limit by nearly five orders of magnitude compared to that of the pure CHA.

Ultrabright red-emitted aggregation-induced luminescence microspheres-based lateral flow immunoassay for furosemide detection in slimming products

Furosemide (Furo) is an effective diuretic drug, and it has been illegally used in slimming dietary supplements. Herein, an anti-Furo monoclonal antibody (anti-Furo mAb) was prepared, and the obtained anti-Furo mAb showed a high binding affinity of 3.48 × 109 L/mol to Furo molecules. Using an ultrabright red-emitted aggregation-induced luminescence microspheres (AIEFMs) as a signal amplification probe, a novel fluorescent lateral flow immunoassay (LFIA) was developed for the rapid and sensitive determination of Furo in slimming products. Under optimal conditions, the proposed AIEFM-LFIA exhibited a high sensitivity to Furo quantitative determination with half-maximal inhibitory concentration and limit of detection at 0.31 and 0.049 ng/mL, respectively. These values were 8.8- and 4.2-fold lower than those of enzyme-linked immunosorbent assay (ELISA). The intra- and inter-assays showed the average recovery of AIEFM-LFIA ranging from 88.71% to 111.78% with coefficient of variation ranging from 2.58% to 13.23%, indicating that the proposed method has an accepted accuracy and precision for furosemide quantitative determination. In addition, the proposed AIEFM-LFIA also showed a good consistency with the ELISA method. Given the signal amplification of AIEFMs and high affinity of the as-prepared mAb, the developed AIEFM-LFIA provides great potential for the sensitive and rapid detection of Furo in slimming products.

An ultra-sensitive one-pot RNA-templated DNA ligation rolling circle amplification-assisted CRISPR/Cas12a detector assay for rapid detection of SARS-CoV-2

Rapid, sensitive, and one-pot diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an extremely important role in point-of-care testing (POCT). Herein, we report an ultra-sensitive and rapid one-pot enzyme-catalyzed rolling circle amplification-assisted CRISPR/FnCas12a assay, termed OPERATOR. OPERATOR employs a single well-designed single-strand padlock DNA, containing a protospacer adjacent motif (PAM) site and a sequence complementary to the target RNA which procedure converts and amplifies genomic RNA to DNA by RNA-templated DNA ligation and multiply-primed rolling circle amplification (MRCA). The MRCA amplicon of single-stranded DNA is cleaved by the FnCas12a/crRNA complex and detected via a fluorescence reader or lateral flow strip. OPERATOR presents outstanding advantages including ultra-sensitivity (1.625 copies per reaction), high specificity (100%), rapid reaction speed (∼30 min), easy operation, low cost, and on-spot visualization. Furthermore, we established a POCT platform by combining OPERATOR with rapid RNA release and a lateral flow strip without professional equipment. The high performance of OPERATOR in SARS-CoV-2 tests was confirmed using both reference materials and clinical samples, and the results suggest that is readily adaptable for point-of-care testing of other RNA viruses.

Highly sensitive fluorescent turn-on lateral flow strip for chlorothalonil based on an indicator displacement ratiometric fluorescent assay

Nanomaterial based indicator displacement assays (IDA) have demonstrated interesting applications in chemosensing, however, restricted to fluorescence quenching nanomaterials. Fluorescent metal nanocluster (NCs) serving as a receptor in IDA may provide new sensing applications. In this work, we utilized GSH/CD-AuNCs as receptor and kaempferol/Al3+ complex (KAE/Al3+) as indicator to fabricate a new nanomaterial based IDA. Due to inner filter effect (IFE) and photoinduced electron transfer (PET) effect, the fluorescence of GSH/CD-AuNCs was greatly quenched by KAE/Al3+ complex. In the presence of chlorothalonil (CHL), CHL was more likely to enter the cavity than KAE/Al3+ due to its higher affinity with β-CD, thereby restoring the AuNCs fluorescence. Meanwhile, the fluorescence of KAE/Al3+ could be greatly enhanced after β-CD inclusion. Based on the above principle, an indicator displacement ratiometric fluorescent assay (IDRFA) for CHL was successfully constructed. Moreover, we integrated this IDRFA with lateral folw assay (LFA) format to develop a novel fluorescent turn-on IDA-LFA for CHL, which facilitated the naked eye discrimination and sensitive quantitative detection through LFA. Due to the advantages of rapid (completed within 1 min), and highly sensitive (even higher than some immunochromatographic strips), this novel IDA-LFA strip have high potential in rapid screening CHL.

Smartphone-Based Quantitative Detection of Ochratoxin A in Wheat via a Lateral Flow Assay.

Ochratoxin A (OTA) poses a severe health risk to livestock along the food chain. Moreover, according to the International Agency for Research on Cancer, it is also categorized as being possibly carcinogenic to humans. The lack of intelligent point-of-care test (POCT) methods restricts its early detection and prevention. This work establishes a smartphone-enabled point-of-care test for OTA detection via a fluorescent lateral flow assay within 6 min. By using a smartphone and portable reader, the assay allows for the recording and sharing of the detection results in a cloud database. This intelligent POCT provided (iPOCT) a linearity range of 0.1-3.0 ng/mL and a limit of detection (LOD) of 0.02 ng/mL (0.32 µg/kg in wheat). By spiking OTA in blank wheat samples, the recoveries were 89.1-120.4%, with a relative standard deviation (RSD) between 3.9-9.1%. The repeatability and reproducibility were 94.2-101.7% and 94.6-103.4%, respectively. This work provides a promising intelligent POCT method for food safety.

NIR-II fluorescent nanoprobe-labeled lateral flow biosensing platform: A high-performance point-of-care testing for carcinoembryonic antigen

Fluorescent lateral flow immunoassay (LFA) is one of the most common analytical platforms for point-of-care testing (POCT), which is capable of facile and early screening of biomarkers. Notably, fluorescent probes play a decisive role in analytical performances of LFA. Herein, we report a novel LFA based on the rare earth doped nanoparticles (RENPs) emitting in the second near-infrared (NIR-II) window for the detection of biomarkers, such as carcinoembryonic antigen (CEA). Benefiting from the dual fluorescent emission at NIR-II window, strong fluorescent penetration, low autofluorescence and excellent photostability of RENPs, this proposed NIR-II LFA displays a good linear relationship ranging from 1 to 320 ng mL−1. The detection limit is as low as 0.37 ng mL−1, which is of 13.5 times lower than the clinical cutoff value. Overall, NIR-II LFA biosensing platform based RENPs not only exhibits high sensitivity, accuracy and specificity, but also have characteristics of rapidity, simplicity and low cost. It holds high potential for early diagnosis of tumor biomarkers in POCT.

Bio-conjugated nanoarchitectonics with dual-labeled nanoparticles for a colorimetric and fluorescent dual-mode serological lateral flow immunoassay sensor in detection of SARS-CoV-2 in clinical samples.

Serological detection of antibodies for diagnosing infectious diseases has advantages in facile diagnostic procedures, thereby contributing to controlling the spread of the pathogen, such as in the recent SARS-CoV-2 pandemic. Lateral flow immunoassay (LFIA) is a representative serological antibody detection method suitable for on-site applications but suffers from low clinical accuracy. To achieve a simple and rapid serological screening as well as the sensitive quantification of antibodies against SARS-CoV-2, a colorimetric and fluorescent dual-mode serological LFIA sensor incorporating metal-enhanced fluorescence (MEF) was developed. For the strong fluorescence signal amplification, fluorophore Cy3 was immobilized onto gold nanoparticles (AuNPs) with size-controllable spacer polyethyleneglycol (PEG) to maintain an optimal distance to induce MEF. The sensor detects the target IgG with a concentration as low as 1 ng mL-1 within 8 minutes. The employment of the MEF into the dual-mode serological LFIA sensor shows a 1000-fold sensitivity improvement compared with that of colorimetric LFIAs. The proposed serological LFIA sensor was tested with 73 clinical samples, showing sensitivity, specificity, and accuracy of 95%, 100%, and 97%, respectively. In conclusion, the dual-mode serological LFIA has great potential for application in diagnosis and an epidemiological survey of vaccine efficacy and immunity status of individuals.

Molecular simulation-guided aptasensor design of robust and sensitive lateral flow strip for cadmium ion detection.

Lateral flow fluorescence strip (LFFS) aptasensor have been widely used for on-site target detection. However, they are limited by low sensitivity and strong background signals owing to the inappropriate design of molecule probes. Herein, we employed molecular simulations to improve the sensitivity of LFFS by the optimization of the DNA probe length and sequence, which is a critical parameter for the competitive approach of the aptasensor. Simulation results revealed that a probe with 30 nt can maximize the hybridization yield of aptamer to reduce the background signal. More importantly, the simulation results highlighted the Cd2+ concentration-dependent conformational changes in the aptamer. It is essential to block its hybridization with a probe, and consequently, yield sensitive and target concentration-dependent fluorescence signal. Considering these results, we developed a sensitive aptamer-based fluorescent lateral flow strip for rapid Cd2+ detection. The fluorescence intensity of this strip exhibited an excellent linear relationship with the Cd2+ concentration ranging from 63 nM to 1000 nM (R2 = 0.9724). The limit of detection was determined to be 30 nM (S/N = 3). This method was also applied for the detection of Cd2+ in river water samples in the range from 92.9 ± 1.0% to 108.6 ± 1.4%. Moreover, the detected concentration in water samples is below the harmful levels (267 nM) recommended by WHO standards in drinking water. The use of molecular simulations is a significant addition to cost and resource-effective aptasensor development protocol, and it can be readily expanded to design aptasensors for other targets.