Colorimetric Readout Research Articles

Gold Nanoparticles Decorated CoAl LDH Monolayer: A Peroxidase-Like Nanozyme for Sensitive Colorimetric Detection of Acetylcholinesterase and Inhibitors.

Monitoring acetylcholinesterase (AChE) activity and its inhibitor is crucial yet challenging for the early diagnosis and treatment of neurological diseases. In this study, we present Au nanoparticle decorated CoAl layered double hydroxide monolayer (Au@CoAl-LDH-m) as a peroxidase-like (POD) nanozyme for the sensitive colorimetric detection of AChE and its inhibitor, thiamine pyrophosphate (TPP). Remarkably, the Au@CoAl-LDH-m nanozyme can catalyze the oxidation of chromogenic substrates through its POD-like activity, which is effectively inhibited by thiocholine (TCh, a catalytic product of AChE), thereby enabling detection of AChE and TPP through a visible colorimetric readout. The approach provides a highly sensitive and specificity assay with a broader linear response range (1-100 mU mL-1 for AChE and 1-1000 ng mL-1 for TPP) and a low detection limit (0.092 mU mL-1 for AChE and 0.201 ng mL-1 for TPP), respectively. These results highlight the significant potential of Au@CoAl-LDH-m for advancing colorimetric sensors in detecting small molecules across various biological applications.

Adaptation of a Model Spike Aptamer for Isothermal Amplification-Based Sensing.

Isothermal amplification (IA) techniques like rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) have gained significant attention in recent years due to their ability to rapidly amplify DNA or RNA targets at a constant temperature without the need for complex thermal cycling equipment. Such technologies, combined with colorimetric systems rendering visual confirmation of the amplification event, are ideal for the development of point-of-need detection methods suitable for field settings where access to specialized laboratory equipment is limited. The utility of these technologies, thus far limited to DNA and RNA targets, could be broadened to a wide range of targets by using aptamers. Composed of DNA or RNA themselves, aptamers can bind to substances, including proteins, metabolites, and inorganic substances. Their nucleic acid nature can potentially allow them to serve as a bridge, extending the reach of DNA/RNA-centric technologies to the broader molecular world. Indeed, the change in aptamer conformation occurring during ligand interaction can be used to elaborate ligand-responding RCA or LAMP templates. By using an existing aptamer targeting SARS-CoV-2 Spike protein as a model, we explored the possibility of establishing ligand-responsive IA systems. Our study used aptamers with simple sequence modifications as templates in LAMP assays and hyperbranched RCA (HRCA) by exploiting the dynamic nature of the model aptamer to trigger these IA systems. Importantly, our work uniquely demonstrates that this aptamer's dynamic response to ligand binding can regulate both RCA and LAMP processes. This novel approach of using aptamer conformational changes to trigger LAMP paves the way for new aptamer-based detection assays. Our system detects 50 nM of Spike protein, with LAMP occurring within 30 min in the presence of Spike. The colorimetric readout showed clear results, allowing for the detection of Spike protein presence.

Benchtop to at-home test: Amplicon-depleted CRISPR-regulated loop mediated amplification at skin-temperature for viral load monitoring

CoRPLA (CRISPR-regulated One-pot Recombinase Polymerase Loop-mediated Amplification) is an amplicon-depleted skin-temperature operated iNAAT designed for at-home testing. It uses specially designed loop primers to enhance isothermal amplification, triggering Cas12 for in-situ amplicon depletion and signal amplification. This method addresses issues like amplicon-derived aerosol contamination and complex assay formats, enabling quantitative detection with sub-attomolar sensitivity (0.5 cps/μL). CoRPLA employs a DNA hydrogel wearable tape for real-time, colorimetric readout, allowing visual differentiation of pathogen loads. It was validated with clinical samples for SARS-CoV-2, RSV, influenza A, and HPV, successfully identifying multi-level viral loads of the positive cases with results consistent with qPCR. Offering high sensitivity while eliminating false positives from aerosol contamination, CoRPLA bridges the molecular assay from benchtop to home for daily viral infections monitoring.

On-Site Visualization Assay for Tumor-Associated miRNAs: Using Ru@TiO2 as a Peroxidase-like Nanozyme.

Accurate diagnosis of highly aggressive and deadly tumors is essential for effective treatment and improved patient outcomes, and microRNAs (miRNAs) have emerged as crucial biomarkers for their roles in tumor initiation, progression, and metastasis. Herein, we present an on-site visualization colorimetric assay for tumor-associated miRNAs using ruthenium nanoparticle decorated titanium dioxide nanoribbon (Ru@TiO2) as a peroxidase-like (POD) nanozyme. Remarkably, the Ru@TiO2 nanozyme can catalyze the oxidation of chromogenic substrates through its POD-like activity, which is effectively inhibited by pyrophosphate generated during the rolling circle amplification process, thereby enabling miRNA detection through a visible colorimetric readout. This approach provides a highly sensitive and specificity assay for miRNAs in diluted human serum with a detection limit of 100 pM. It shows great potential for clinical diagnostics and biological research, offering a promising tool for early cancer diagnosis and molecular diagnostics.

A target-triggered colorimetric sensor for ultrasensitive detection of miRNAs based on self-powered three-dimensional DNA walker

MicroRNAs (miRNAs) play an important role in the process of heart failure (HF) and are emerging biomarkers that can be used for the auxiliary diagnosis of HF. However, it is very challenging to accurately analyze the expression levels of trace miRNAs in complex clinical samples. Here, we developed an enzyme-free colorimetric sensor for the ultrasensitive detection of miRNA-423-5p (HF-associated miRNA) based on three-dimensional DNA walkers constructed from functional nucleic acids and gold nanoparticles (AuNPs). DNAzyme with cleavage activity was specifically activated by miRNA-423-5p to sustainably cleave the substrate, thereby releasing the trigger sequence to initiate the subsequent mismatched catalytic hairpin assembly (MCHA) cycle. Then, as the MCHA cycle proceeded to continuously expose the G-quadruplex (GQ) sequence, the sequence bound with hemin to form a large amount of GQ/hemin DNAzyme on the surface of the AuNPs, which rapidly catalyzed the chromogenic oxidation of 3,3′,5,5′-tetramethylbenzidine to yield an amplified colorimetric signal readout. The colorimetric sensor exhibited an ultralow detection limit (32 fM), showed excellent specificity and performed well in serum samples. The sensor was applied to detect miRNA-423-5p in clinical plasma samples from healthy individuals and HF patients, and the results revealed its good clinical application in HF diagnosis. Thus, the developed colorimetric sensor provides a convenient detection tool for early screening and diagnosis of HF, as well as for pathophysiological studies.

An at-home and electro-free COVID-19 rapid test based on colorimetric RT-LAMP.

In the fight against virus-caused pandemics like COVID-19, diagnostic tests based on RT-qPCR are essential, but they are sometimes limited by their dependence on expensive, specialized equipment and skilled personnel. Consequently, an alternative nucleic acid detection technique that overcomes these restrictions, called loop-mediated isothermal amplification following reverse transcription (RT-LAMP), has been broadly investigated. Nevertheless, the developed RT-LAMP assays for SARS-CoV-2 detection still require laboratory devices and electrical power, limiting their widespread use as rapid home tests. This work developed a flexible RT-LAMP assay that gets beyond the drawbacks of the available isothermal LAMP-based SARS-CoV-2 detection, establishing a simple and effective at-home diagnostic tool for COVID-19. A multiplex direct RT-LAMP assay, modified from the previously developed test was applied to simultaneously identify the two genes of SARS-CoV-2. We used a colorimetric readout, lyophilized reagents, and benchmarked an electro-free and micropipette-free method that enables sensitive and specific detection of SARS-CoV-2 in home settings. Forty-one nasopharyngeal swab samples were tested using the developed home-testing RT-LAMP (HT-LAMP) assay, showing 100% agreement with the RT-qPCR results. This is the first electrically independent RT-LAMP assay successfully developed for SARS-CoV-2 detection in a home setting. Our HT-LAMP assay is thus an important development for diagnosing COVID-19 or any other infectious pandemic on a population scale.

Janus-structured colorimetric and fluorescent nanocomposites with highly luminescent retention to enhance point-of-care diagnosis

Lateral flow immunoassay (LFIA) with dual-readout signals offers significant advantages in point-of-care testing due to its adaptability to various analysis scenarios. However, colorimetric and fluorescent nanocomposites (cf-NCs) composed of plasmonic and luminescent components often suffer from low luminescent efficiency because of severe optical interference between the two functional components. Herein, we propose a simple self-assembly strategy for fabricating novel cf-NCs with a Janus structure (J-cf-NCs) using oleylamine ligand-coated gold nanoparticles (OA-AuNPs) and aggregation-induced emission luminogens (AIEgens) as building blocks. For comparison, two other cf-NCs with core–shell (CS-cf-NCs) and co-embedding (CE-cf-NCs) structures were synthesized to explore the effects of spatial intersection of OA-AuNPs and AIEgens on their optical properties. Our results demonstrate that J-cf-NCs exhibit the highest fluorescence retention (75 %), as the face-to-face spatial distribution of OA-AuNPs and AIEgens effectively prevents fluorescence resonance energy transfer and minimizes inner-filter effect-induced fluorescence quenching. When integrated with the LFIA platform, the developed J-cf-NC-LFIA demonstrated excellent detection performance for both qualitative and quantitative determination of various target analytes in sandwich and competitive formats. Specifically, the visual limit of detection (vLOD) for C-reactive protein using J-cf-NC-LFIA showed approximately an eight-fold improvement compared to the classical colloidal gold test strip, while the quantitative sensitivity was two orders of magnitude greater than that of colorimetric readout. Furthermore, the vLOD for aflatoxin M1 in milk samples was 0.5 ng⋅mL–1, with a quantitative sensitivity 38-fold higher than that of the colorimetric pattern. Overall, the proposed J-cf-NCs exhibit significant potential for enhancing the detection performance of LFIA platforms.

Pushing the Limits of Lateral Flow Immunoassay by Digital SERS for the Ultralow Detection of SARS‐CoV‐2 Virus

Lateral flow immunoassays (LFIAs) are easy‐to‐use antigen tests that provide different signal readouts, with colorimetric readouts being the most commonly used. However, these analytical devices have relatively low sensitivity and produce semiquantitative results, limiting their diagnostic applications. Herein, we address these challenges by implementing a digital surface‐enhanced Raman spectroscopy (SERS)‐based LFIA for the accurate and ultrasensitive quantitative detection of SARS‐CoV‐2 nucleocapsid (N) protein. Compared with average SERS intensity measurements, the digital approach allowed to overcome fluctuations in Raman scattering signals, thereby increasing the analytical sensitivity of the assay. Our method exhibited a quantification range of the viral protein in nasal swabs from 0.001 to 10 pg mL−1, and a limit of detection down to 1.9 aM (0.9 fg mL−1), improving colorimetric LFIAs and conventional‐SERS‐based LFIAs by several orders of magnitude. Importantly, this approach shows an analytical sensitivity of 0.03 TCID50 mL−1, which is greater than that reported by other immunoassays. In conclusion, we successfully demonstrate the robust detection and quantification of SARS‐CoV‐2N protein in nasal swabs at ultralow concentrations. The improvement in the sensitivity of LFIA by digital SERS may pave the way to translate this technology into the diagnostic arena for the ultrasensitive detection of microbes and disease biomarkers.

A modular cell-free protein biosensor platform using split T7 RNA polymerase.

Conventional laboratory protein detection techniques are not suitable for point-of-care (POC) use because they require expensive equipment and laborious protocols, and existing POC assays suffer from long development timescales. Here, we describe a modular cell-free biosensing platform for generalizable protein detection that we call TLISA (T7 RNA polymerase-Linked ImmunoSensing Assay), designed for extreme flexibility and equipment-free use. TLISA uses a split T7 RNA polymerase fused to affinity domains against a protein. The target antigen drives polymerase reassembly, inducing reporter expression. We characterize the platform, then demonstrate its modularity by using 16 affinity domains against four different antigens with minimal protocol optimization. We show TLISA is suitable for POC use by sensing human biomarkers in serum and saliva with a colorimetric readout within one hour and by demonstrating functionality after lyophilization. Altogether, this technology could have potentially revolutionary impacts, enabling truly rapid, reconfigurable, equipment-free detection of virtually any protein.

Semiquantitative Paper-Based Microfluidic Surrogate Virus Neutralization Test for SARS-CoV-2 Neutralizing Antibodies.

Neutralizing antibodies (nAbs) produced from infection or vaccination play an important role in acquired immunity. Determining virus-specific nAb titers is a useful tool for measuring aquired immunity in an individual. The standard methods to do so rely on titrating serum samples against live virus and monitoring viral infection in cultured cells which requires high biosafety level containment. The surrogate virus neutralization test (sVNT) reduces the biohazards and it is suitable for designing rapid test device in a lateral flow assay (LFA) format. Here, we introduce the fabrication and development of a unique paper-based LFA device for determining the level of SARS-CoV-2 nAb in a sample with a semiquantitative direct colorimetric readout. A LFA-based gradient assay design was used to facilitate the sVNT, where the spike glycoprotein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) stand in as proxies for viruses and cells, respectively. The gradient assay employed multiple test dots of ACE2 spotted in increasing concentration along the sample flow path and gold nanoparticle-conjugated RBD for readout. In this way, the number of developed spots is inversely proportional to the concentration of nAbs present in the sample. The assay was tested with both standard solutions of nAb as well as human serum samples. We have demonstrated that the device can effectively provide semiquantitative test results of nAbs by direct instrument-free colorimetric detection.

A portable smartphone detection of ctDNA using MnB2 nanozyme and paper-based analytical device

The development of point-of-care testing (POCT) for circulating tumor DNA (ctDNA) is meaningful for the non-invasive cancers screening and diagnosis, particularly in resource-limited settings. The microfluidic paper-based analytical device (μPAD) provides an ideal platform, its application in ctDNA assays remains underexplored. In this work, a multifunctional μPAD was manufactured, which can enhance the efficiency and reduce the cost of ctDNA sensing. Additionally, a smartphone-based application analysis was fabricated for convenient, portable detection and colorimetric signal readout. Moreover, the novel oxidase-like MnB2 nanozyme was introduced in the sandwiches sensing strategy, utilizing its catalytic properties to effectively generate a colorimetric signal. The use of MnB2 nanozyme in sensing application is relatively novel, and its catalytic performance and mechanism was thoroughly evaluated via experiment and density functional theory (DFT) calculations. After optimizing the detection conditions, the proposed biosensor exhibited satisfactory results. Furthermore, the method was successfully used to detect ctDNA in tumor cell lysates and peripheral blood samples from tumor-bearing mice. The results were consistent with standard qPCR method, affirming the reliability of our POCT analysis device in ctDNA detection. Thus, this work not only provides a paper-based POCT device and intelligent analysis tool for portable cancers diagnosis, but it also paves a new application path for MnB2 nanozyme in the sensing filed.

Target Recognition Triggered Split DNAzyme based Colorimetric Assay for Direct and Sensitive Methicillin-Resistance Analysis of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) holds significant clinical importance. This work presents a new method for detecting methicillin-resistant Staphylococcus aureus (S. aureus) in clinical samples. The method uses an aptamer-based colorimetric assay that combines a recognizing probe to identify the target and split DNAzyme to amplify the signal, resulting in a highly sensitive and direct analysis of methicillin-resistance. The identification of the PBP2a protein on the membrane of S. aureus in clinical samples leads to the allosterism of the recognizing probe, and thus provides a template for the proximity ligation of split DNAzyme. The proximity ligation of split DNAzyme forms an intact DNAzyme to identify the loop section in the L probe and generates a nicking site to release the loop sequence ("3" and "4" fragments). The "3" and "4" fragments forms an intact sequence to induce the catalytic hairpin assembly, exposing the G-rich section. The released the G-rich sequence of LR probe induces the formation of G-quadruplex-hemin DNAzyme as a colorimetric signal readout. The absorption intensity demonstrated a strong linear association with the logarithm of the S. aureus concentration across a wide range of 5 orders of magnitude dynamic range under the optimized experimental parameters. The limit of detection was calculated to be 23 CFU/ml and the method showed high selectivity for MRSA.

Photonic Nanochains for Continuous Glucose Monitoring in Physiological Environment.

Diabetes is a common disease that seriously endangers human health. Continuous glucose monitoring (CGM) is important for the prevention and treatment of diabetes. Glucose-sensing photonic nanochains (PNCs) have the advantages of naked-eye colorimetric readouts, short response time and noninvasive detection of diabetes, showing immense potential in CGM systems. However, the developed PNCs cannot disperse in physiological environment at the pH of 7.4 because of their poor hydrophilicity. In this study, we report a new kind of PNCs that can continuously and reversibly detect the concentration of glucose (Cg) in physiological environment at the pH of 7.4. Polyacrylic acid (PAA) added to the preparation of PNCs forms hydrogen bonds with polyvinylpyrrolidone (PVP) in Fe3O4@PVP colloidal nanoparticles and the hydrophilic monomer N-2-hydroxyethyl acrylamide (HEAAm), which increases the content of PHEAAm in the polymer shell of prepared PNCs. Moreover, 4-(2-acrylamidoethylcarbamoyl)-3-fluorophenylboronic acid (AFPBA), with a relatively low pKa value, is used as the glucose-sensing monomer to further improve the hydrophilicity and glucose-sensing performances of PNCs. The obtained Fe3O4@(PVP-PAA)@poly(AFPBA-co-HEAAm) PNCs disperse in artificial serum and change color from yellow-green to red when Cg increases from 3.9 mM to 11.4 mM, showing application potential for straightforward CGM.

Photocatalytic colorimetry and fluorescence dual-signal biosensor for instant determination of terminal deoxynucleotidyl transferase

Terminal deoxynucleotidyl transferase (TdT) is a crucial biomarker for diseases such as lymphoblastic leukemia. Herein, a photocatalytic colorimetry and fluorescence dual-signal biosensor (PhoCoF-sensor) for simple, low-cost and sensitive detection of TdT was developed, which integrated with the TdT-triggered polymerization reaction and the fluorescence-photocatalytic dual performance of double-strand DNA/SYBR Green-I (dsDNA/SG-I) complex. The target TdT catalyzed the elongation of single-stranded DNA in the dTTP substrate pool, and then hybridized with complementary DNA to form dsDNA/SG-I complex that exhibited obvious fluorescent and excellent photocatalytic activity. Reactive oxygen species were generated from photosensitization of the dsDNA/SG-I complex acted as the visual colorimetric readout. The PhoCoF-sensor was successfully used to detect TdT activity with a series of color and fluorescence changes in the concentration range from 0.5 U/mL to 20.0 U/mL. The PhoCoF-sensor further improves the assay simplicity and accuracy and reduces the time and cost, which overcomes the limitations of traditional detection TdT methods and single-signal sensors. Additionally, this PhoCoF-sensor can readily be integrated with centrifugal microfluidic device for one-step test and achieve quantitative determination without sophisticated instruments. Moreover, the PhoCoF-sensor has good sensitivity and good practical reliability and is suitable for instant determination in early disease diagnosis and drug discovery.

Rapid and sensitive aptamer-linked immunosorbent assay with aptamer-templated silver nanoparticles for detection of aflatoxin B1 in medicinal and edible food

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin commonly found in various medicinal and edible foods (MEFs). AFB1 in MEFs poses an inevitable threat to human health. High-performance analytical techniques are required for AFB1 detection in the quality assessment of MEFs. This work reports a novel aptamer-linked immunosorbent assay (ALISA) using aptamer-templated silver nanoparticles (AgNPs) as signal transducers for AFB1 detection. The effect of aptamer on the morphology and properties of AgNPs was systematically investigated. Aptamers with lower concentrations can induce the aggregation of AgNPs, thereby exhibiting excellent photothermal effects. Whereas, aptamers with higher concentrations mediate small-sized AgNPs' generation, demonstrating outstanding extinction coefficients. Accordingly, the effect of aptamer on the properties of AgNPs was used to construct a novel ALISA. Specifically, AFB1 aptamers were immobilized on 96-well plates via AFB1 and antibodies. In the presence of sodium borohydride (NaBH4) and silver nitrate (AgNO3), AgNPs were synthesized in a one-pot manner using the free aptamer as a template. The concentration of free aptamer can regulate AgNP morphology, affecting extinction coefficient and photothermal effect, enabling visual colorimetric and portable photothermal signal readout. When employed for AFB1 detection in MEFs, the proposed ALISA demonstrated rapid detection speed and high sensitivity, with a response time of 8 min and a colorimetric signal detection limit of 0.33 pg/mL. We expect that this ALISA not only enables rapid and cost-effective AFB1 detection but also offers a universal, user-friendly, and highly sensitive method for detecting other MEF hazards.

Synergistic synthesis of gold nanoflowers as upconversion near-infrared nanoprobe energy acceptor and recognition unit for improved hydrogen sulfide sensing

A highly sensitive and selective upconversion near-infrared (NIR) fluorescence and colorimetric dual readout hydrogen sulfide (H2S) nanoprobe was constructed based on the excellent NIR fluorescence emission performance of upconversion nanomaterials (UCNPs), the specific recognition effect of synergistically synthesized gold nanoflowers (trypsin-stabled AuNFs (Try-AuNFs)) and the effective NIR fluorescence quenching capability. In this assay, the sensing strategy included three processes. First of all, the synthesized UCNPs can emit 803 nm NIR fluorescence when they were excited by 980 nm excitation light. Secondly, as a result of the principle of fluorescence resonance energy transfer (FRET), Try-AuNFs can effectively quench the NIR fluorescence of UCNPs at 803 nm, which can effectively improve the signal-to-background ratio of nanoprobes, thereby improving the sensitivity of the probes. Thirdly, in the presence of H2S, the Try protective layer on the surface of Try-AuNFs was specifically penetrated, which will subsequently cleave Try-AuNFs via the strong S–Au bond. As such, the NIR fluorescence of UCNPs will be restored, achieving high selectivity and sensitivity detection of H2S. Under optimized conditions, the linear response range of H2S was 0.1–300 μM, and the detection limit was 53 nM. It is worth noting that the Try on the surface of Try-AuNFs via the synergistic effect can increase the steric hindrance of the probe, and this can effectively prevent the interaction between the probe with biothiols (cysteine (Cys), homocysteine (Hcy)) and other natural amino acids (non-thiol-containing) with resultant in the high selectivity regarding the detection of H2S in human serum, which is unlikely to be achieved by AuNFs synthesized by the gold seed method (Se-AuNFs). This work not only provided a new type of UCNPs fluorescence quencher and recognition unit, but also exemplified that the use of the physical properties (steric hindrance) of protein ligands on the surface of nanoflowers can improve the specificity of the probe. This will provide new ideas for the design of other nanoprobes.

Colorimetric analysis platform based on thin layer chromatography for monitoring gluten cross-contamination in food industry

Monitoring of the accidental presence of gluten (Glu), resulting from cross-contamination, is imperative in different industries, in particular food industry. The objective of this study was the development of an analytical platform utilizing thin-layer chromatography (TLC) with colorimetric read-out for making binary (yes/no) decisions on surfaces and/or point of these industries. The composition of the extractive phase was optimized with commercial products used in cleaning processing lines. Subsequently, an exploration of TLC separation and detection was undertaken. CN-modified nanosilica plates and 30:70 acetonitrile:water were used to achieve a selective signal for Glu residues. The study of the detection performance showed that both spectroscopic measurement and image analysis were resulted in satisfactory results for quantitate analysis (RSD = 5 %, LOD = 0.12 mg). The practical application of the proposed methodology on surfaces of the food processing lines. This work demonstrated the operational feasibility in detecting gluten cross-contaminations within the food processing industry.

Construction of a Colorimetric and Fluorescence Dual-Mode Immunoassay Detection of Alpha-Hemolysin in Milk.

Alpha-hemolysin (Hla) is a major virulence factor secreted by Staphylococcus aureus (S. aureus), which can lyse a variety of mammalian cells and help bacteria evade the host immune system or antibiotics, posing a safety hazard to human health. Therefore, it is critical to establish a quick-responsive and sensitive method for Hla detection to ensure food safety. In this work, a dual-mode immunoassay was developed with both colorimetric and fluorescent readouts for discriminative detection of Hla. The proposed sensing system consists of p-phenylenediamine (PPD) and fluorescein, where fluorescein functions as a fluorescent reporter, and PPD serves a dual function as a colorimetric reporter and fluorescence quencher. Subsequently, the reaction system of this method was optimized, and the detection limit, sensitivity, and specificity were evaluated. Under optimal conditions, the proposed method possesses excellent analytical performance in the range from 0.5 to 500 ng/mL with a limit of detection as low as 0.5 ng/mL. Noteworthy, this method was successfully employed for the detection of Hla in milk with good selectivity and high accuracy. Overall, the dual-mode immunoassay provides a superior platform for the on-site, quantitative, and accurate detection of Hla in food samples.

Towards rapid colorimetric detection of extracellular vesicles using optofluidics-enhanced color-changing optical metasurface.

Efficient transportation and delivery of analytes to the surface of optical sensors are crucial for overcoming limitations in diffusion-limited transport and analyte sensing. In this study, we propose a novel approach that combines metasurface optics with optofluidics-enabled active transport of extracellular vesicles (EVs). By leveraging this combination, we show that we can rapidly capture EVs and detect their adsorption through a color change generated by a specially designed optical metasurface that produces structural colors. Our results demonstrate that the integration of optofluidics and metasurface optics enables spectrometer-less and label-free colorimetric read-out for EV concentrations as low as 107 EVs/ml, achieved within a short incubation time of two minutes.

Quantification of creatinine in whole blood by a paper-based device using an RGB sensor.

Creatinine, an important biomarker for renal functions, is often conventionally estimated using the gold standard Jaffe reaction from blood, which involves the usage of a spectrophotometric measurement, thus restricting its utilization scope in point-of-care settings. Here, we report the development of a method for the single-step quantification of creatinine from whole blood using a paper-based microfluidic device. Our platform uses Whatman filter paper integrated with an LF1 membrane. The on-chip separation of blood plasma is achieved through the LF1 membrane, while the Whatman component of the device contains the embedded reagents for the Jaffe reaction. The combination of two different grades of paper enables a single-step quantification of creatinine as the separated blood plasma traverses to the reaction zone through capillary imbibition. Colorimetric readouts were quantified using an RGB sensor instead of a smartphone, which is highly platform dependent and incurs a relatively higher cost compared to the other components in typical point-of care (POC) devices. Our sensor was integrated within a 3D box, thereby making the detection virtually instrument free and perfectly suited for POC settings. The limit of detection (LOD) of our device was 0.219 mg dL-1, which falls within the lower range of physiological values. The coefficient of determination (R2) for the linearity and median accuracy were 0.978 and 94.047%, respectively. The relative standard deviation (RSD) for precision measurements remained below 5% for the developed protocol. Furthermore, we validated the performance of our device with 35 clinical samples in laboratory settings against the gold standard measurements. Our Bland-Altman plot as well as t-test and chi-square test results clearly confirmed the validity of our device within a 95% confidence interval.