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A new analytical method for chronic kidney disease (CKD) detection utilizing paper spray mass spectrometry (PS-MS) combined with machine learning is presented. The analytical protocol is rapid and simple, based on metabolic profile alterations in urine. Anonymized raw urine samples were deposited (10 μL each) onto pointed PS-MS sample strips. Without waiting for the sample to dry, 75 μL of acetonitrile and high voltage were applied to the strips, using high resolution mass spectrometry measurement (15 s per sample) with polarity switching to detect a wide range of metabolites. Random forest machine learning was used to classify the resulting data. The diagnostic performance for the potential diagnosis of CKD was evaluated for accuracy, sensitivity, and specificity, achieving results >96% for the training data and >91% for validation and test data sets. Metabolites selected by the classification model as up- or down-regulated in healthy or CKD samples were tentatively identified and in agreement with previously reported literature. The potential utilization of this approach to discriminate albuminuria categories (normo, micro, and macroalbuminuria) was also demonstrated. This study indicates that PS-MS combined with machine learning has the potential to be used as a rapid and simple diagnostic tool for CKD.

A simple, rapid chemical coating and patterning method was developed and optimized for paper-based substrates for use in paper spray mass spectrometry (PS-MS). A variety of chlorosilanes were explored for coating paper substrates, and their effectiveness in forming hydrophobic surfaces was characterized via contact angle goniometry, scanning electron microscopy, and energy dispersive X-ray spectroscopy. Trichloromethylsilane was selected as the primary coating agent because of the short time required to produce a hydrophobic surface (contact angle > 130°), as well as the ease of patterning. Patterning was performed using 3D-printed masks and an oxygen/plasma cleaner. Optimal mask thickness and oxygen/plasma cleaning parameters were determined to produce channels varying from 0.5 to 2.5 mm in width. The effectiveness of the patterned substrates for PS-MS was determined via analysis of four antiretrovirals: emtricitabine, lamivudine, efavirenz, and dolutegravir. Calibration curves were made for each antiretroviral at varying channel widths, and the limits of detection and limits of quantification for each drug were determined. These results show that this patterning method results in an average 7.2-fold improvement in sensitivity and an average 190-fold improvement in limits of detection over uncoated paper substrates in a neat matrix. In a proof-of-concept experiment, calibration curves were generated for each antiretroviral in urine. A patterned paper substrate with a 2-mm channel resulted in an average 7.4-fold improvement in sensitivity and an average 18-fold improvement in limits of detection over uncoated paper substrates.

A new method utilizing fluorescent ratiometry is proposed for detecting putrescine and spermidine. The method involves the use of a fluorescent probe comprising a 2D halide perovskite synthesized from octadecylamine-iodine and PbI 2 via a grinding-sonicating technique, along with a Eu 3+ -complex. Upon excitation at 290 nm, the probe fluoresces at two distinguishable wavelengths. The addition of putrescine and spermidine significantly decreases the emission of the 2D halide perovskite at 496 nm, while the emission of the Eu 3+ -complex at 618 nm remains stable. The color changes of the probe depend on the concentration of putrescine and spermidine, and the assay offers linearity over a wide concentration range (30-4000 ng mL -1 ), a low detection limit (4 ng mL -1 for spermidine), and a quick response time. Furthermore, a portable device based on a smartphone can be used to record the color change of the paper test strip using the prepared fluorescent materials. The fluorescence quenching mechanism of the probe is explained as dynamic quenching.-1 for spermidine), and a quick response time. Furthermore, a portable device based on a smartphone can be used to record the color change of the paper test strip using the prepared fluorescent materials. The fluorescence quenching mechanism of the probe is explained as dynamic quenching.

Paper-based electrochemical sensors have the characteristics of flexibility, biocompatibility, environmental protection, low cost, wide availability, and hydropathy, which make them very suitable for the development and application of biological detection. This work proposes electrospun cellulose acetate nanofiber (CA NF)-decorated paper-based screen-printed (PBSP) electrode electrochemical sensors. The CA NFs were directly collected on the PBSP electrode through an electrospinning technique at an optimized voltage of 16 kV for 10 min. The sensor was functionalized with different bio-sensitive materials for detecting different targets, and its sensing capability was evaluated by CV, DPV, and chronoamperometry methods. The test results demonstrated that the CA NFs enhanced the detection sensitivity of the PBSP electrode, and the sensor showed good stability, repeatability, and specificity ( p < 0.01, N = 3). The electrochemical sensing of the CA NF-decorated PBSP electrode exhibited a short detection duration of ∼5-7 min and detection ranges of 1 nmol mL -1 -100 μmol mL -1 , 100 fg mL -1 -10 μg mL -1 , and 1.5 × 10 2 -10 6 CFU mL -1 and limits of detection of 0.71 nmol mL -1 , 89.1 fg mL -1 , and 30 CFU mL -1 for glucose, Ag85B protein, and E. coli O157:H7 , respectively. These CA NF-decorated PBSP sensors can be used as a general electrochemical tool to detect, for example, organic substances, proteins, and bacteria, which are expected to achieve point-of-care testing of pathogenic microorganisms and have wide application prospects in biomedicine, clinical diagnosis, environmental monitoring, and food safety.

Lab-on-a-paper-based devices are promising alternatives to the existing arduous techniques for point-of-need monitoring. The present work reports an instant and facile method to produce a microfluidic paper-based analytical device (μPAD). The fabricated μPAD has been used to detect hypochlorite (OCl - ) by incorporating newly synthesized chromo-fluorogenic ratiometric probes 1 and 2 into the sample reception zone. The probes showed high selectivity and fast response (<10 s) toward OCl - with an excellent linear relationship in the concentration range of 0-100 μM. The concentration-dependent fluorometric change driven by the reaction of 1@μPAD with OCl - has been monitored using gel-doc imaging systems, which is unprecedented. Digitizing the intensity of the colour solution with different mathematical models of colour has developed a straightforward method for monitoring OCl - without any interference from its competitors. 1@μPAD can detect OCl - at ∼10 times lower than the WHO recommended limit. The detection limit of 1@μPAD via a digital camera-based fluorescence technique was found to be better over digital camera-based cuvette assays. Therefore, 1@μPAD has been successfully utilized to monitor OCl - in actual environmental water samples with portability, ease of use, and sensitivity. The analytical RSD was found to be ≤3% based on fluorimetric detection using 1@μPAD. The chemodosimetric reaction between OCl - and the probe was evidenced by UV-vis and fluorescence spectroscopy, 1 H NMR, and ESI-MS. The rapid response time, biocompatibility, low cytotoxicity, 100% aqueous solubility, ratiometric feature, and exclusive OCl - selectivity over other competitive ROS/RNS successfully lead to the application of the probes for bioimaging of exogenous as well as endogenous OCl - in normal cells (HEK293) and cancerous cells (HeLa).

MicroRNAs (miRNAs) have emerged as a promising class of biomarkers for early detection of various cancers, including ovarian cancer. However, quantifying miRNAs in human blood samples is challenging owing to the issues of sensitivity and specificity. In this study, hsa-miR-200a-3p of the miR-200a sub-family, which is a biomarker of ovarian cancer, was used as the analyte to demonstrate the analytical capability of an integrated biosensing platform using an extremely sensitive surface-enhanced Raman scattering (SERS) nanotag-nanoaggregate-embedded beads (NAEBs), magnetic nanoparticles (MNPs), a pair of highly specific locked nucleic acid (LNA) probes, and a semi-automated paper-based electrowetting-on-dielectric (pEWOD) device to provide labor-less and thorough sample cleanup and recovery. A sandwich approach where NAEBs are modified by one LNA-1 probe and MNPs are modified by another LNA-2 probe was applied. Then, the target analyte miRNA-200a-3p was introduced to form a sandwich nanocomplex through hybridization with the pair of LNA probes. The pEWOD device was used to achieve short cleanup time and good recovery of the nanocomplex, bringing the total analysis time to less than 30 min. The detection limit of this approach can reach 0.26 fM through SERS detection. The versatility of this method without the need for RNA extraction from clinical samples is expected to have good potential in detecting other miRNAs.

Paper-based ratiometric fluorescence sensors are normally prepared using two or more types of fluorescent materials on a paper chip for simple, low-cost and fast detection. However, the choice of multi-step and one-step modifications on the paper chip affects the analytical performance. Herein, a novel paper-based dual-emission ratiometric fluorescence sensor was designed for the selective detection of tetracycline (TC). Carbon dots (CDs) modified with Eu 3+ were combined with a sealed paper-based microfluidic chip by two methods: one-step grafting of CDs-Eu 3+ on paper and step-by-step grafting of CDs and Eu 3+ on paper. The analytical performance was studied and optimized respectively. The red fluorescence of Eu 3+ at 450 nm is enhanced and the blue fluorescence of CDs at 617 nm is quenched by energy transfer in the presence of TC. Under optimal conditions, TC is selectively determined in the linear range from 0.1 μM to 100 μM with a detection limit of 0.03 μM by the step-by-step grafting method. In addition, the sealed paper chip could effectively prevent pollution and volatilization from the reagent. This technique has been used to analyze TC in seafood aquaculture water with satisfactory results.

Exosomal microRNAs (miRNAs) play a pivotal role in intercellular communication, regulating gene expression in target cells, and hold significant promise as cancer biomarkers for early detection and screening. However, achieving precise and viable detection of exosomal miRNAs remains a challenge. This paper proposes an all-in-one detection strategy for breast cancer-derived exosomal miRNA-21 on a pen-based paper chip (PPC). The PPC is constructed using a modified automatic pen and lateral flow assay (LFA), which results in a cost-effective fabrication process. The user only needs to add the sample and trigger the top of the self-contained PPC after a period of time to complete the entire detection process. To enhance the sensitivity of exosomal miRNA testing, an enzyme-free catalyzed hairpin assembly (CHA) is further introduced, enabling highly sensitive detection of miRNA-21 with a limit of detection (LOD) of 25 fmol. Additionally, the detection of miRNAs in differentially-expressed cells and clinical samples has also been successfully achieved with high specificity. Overall, the proposed PPC provides an effective tool for detecting early cancer, monitoring diseases, and establishing point of care testing (POCT).

Albuminuria is a clinical condition associated with poor kidney function, diagnosed by determining the ratio of albumin to creatinine concentrations in patient urine samples. We present a high-throughput paper spray mass spectrometry (PS-MS) method for simultaneous quantitation of urinary albumin and creatinine for potential diagnosis of albuminuria. Minimal (urine dilution) or no sample preparation is required. The analytical performance of the method was evaluated, achieving linear calibration curves ( R 2 > 0.99) with little inter-day variability in the slope ( N = 5 days), exhibiting coefficient of variation (CV) of 8% and 3% for albumin and creatinine, respectively. LOD and LOQ for albumin were 2.1 and 7.0 mg L -1 , and for creatinine were 0.01 and 0.03 mmol L -1 , respectively. Intra- and inter-day ( N = 5) precisions (%CV) and accuracies (%bias) were <10% and ±11%, respectively, for both analytes. The method was applied to determine albumin-to-creatinine ratios in anonymous human patient urine samples ( N = 56), and a correlation of R 2 = 0.9744 was achieved between the PS-MS results and validated clinical method results. This work demonstrates the utility of PS-MS to simultaneously quantify a large (albumin) and a small (creatinine) molecule directly in patient urine samples, and its potential as a tool for clinical albuminuria diagnostics.

Fast detection of contaminants of emerging concern (CECs) in water resources is of great environmental interest. Ideally, sustainable materials should be used in water quality monitoring technologies implemented for such purposes. In this regard, the application of bio-based materials aimed at the fabrication of analytical platforms has become of great importance. This research merges both endeavors by exploring the application of chitosan-coated paper, decorated with silver nanoparticles (AgNPs), on surface-enhanced Raman scattering (SERS) spectroscopy studies of two distinct types of CECs dissolved in aqueous samples: an antibiotic (ciprofloxacin) and a pesticide (thiram). Our results indicate the superior SERS performance of biocoated substrates compared to their non-coated paper counterparts. The detection limits achieved for thiram and ciprofloxacin using the biocoated substrates were 0.024 ppm and 7.7 ppm, respectively. The efficient detection of both analytes is interpreted in terms of the role of the biopolymer in promoting AgNPs assemblies that result in local regions of enhanced SERS activity. Taking advantage of these observations, we use confocal Raman microscopy to obtain Raman images of the substrates using ciprofloxacin and thiram as molecular probes. We also demonstrate that these biobased substrates can be promising for on-site analysis when used in conjunction with portable Raman instruments.

Based on surface biomolecular imprinting technology, a rotary microfluidic electrochemical paper-based chip (MIP-ePADs) was proposed for sensitive and selective detection of human interleukin 6 (IL-6) and procalcitonin (PCT). Compared with the traditional method, the sample can be added directly on the MIP-ePAD by rotating the working electrode, which avoids the loss of the liquid to be tested and greatly simplifies the process of electropolymerization imprinting and template elution. Our experimental results show that linear concentration ranges of IL-6 and PCT in the electrochemical molecularly imprinted microfluidic paper-based chip ranged from 0.01 to 5 ng mL -1 , with their detection limits being 3.5 and 2.1 pg mL -1 , respectively. For the detection of actual serum samples, there was no significant difference between the results of MIP-ePADs and the traditional electrochemiluminescence method used in hospitals, indicating that the paper-based chip can be used for stable and accurate analysis and detection. The chip greatly reduces the cost of clinical trials due to its advantages of easy preparation and low cost. The chip can be used for the analysis of non-antibody inflammation markers and can be widely used in home and hospital treatment detection. This method will not only play an important role in rapid detection, but also provide new ideas for the improvement of rapid detection technology.

With growing environmental concern and supply chain uncertainty, now is a fitting time to re-evaluate solvent-free methodologies in forensic chemistry processes. Here, this paper reviews solvent-free approaches for fingermark visualisation, including chemical fuming and vapour phase treatments, dry-transfer treatments, application of heat, and thermal paper specific treatments. After providing historical context, three objectives have been emphasised: identify feasible scenarios for implementing solvent-free methods; showcase the effectiveness of solvent-free methods relative to their nearest solution-based equivalent; and estimate the technological readiness level of each method discussed. Having reviewed the literature, dry-transfer methods of developing latent fingermarks on paper were found to be the most promising and feasible solvent-free approaches for near-term implementation. Such methods make use of standard materials and equipment commonly found in forensic laboratories, are effective at fingermark visualisation, and mitigate most of the pressing issues pertaining to environmental concern and solvent scarcity.

Paracetamol overdose is a leading cause of acute liver failure that can prove fatal. Establishing paracetamol concentration accurately and quickly is critical. Current detection methods are invasive, time-consuming and/or expensive. Non-invasive, rapid and cost-effective techniques are urgently required. To address this challenge, a novel approach, called Paper-Arrow Mass Spectrometry (PA-MS) has been developed. This technique combines sample collection, extraction, enrichment, separation and ionisation onto a single paper strip, and the entire analysis process, from sample to result, can be carried out in less than 10 min requiring only 2 μL of raw human saliva. PA-MS achieved a LOQ of 185 ng mL -1 , mean recovery of 107 ± 7%, mean accuracy of 11 ± 8% and precision ≤5% using four concentrations, and had excellent linearity ( r 2 = 0.9988) in the range of 0.2-200 μg mL -1 covering the treatment concentration range, surpassing the best-in-class methods currently available for paracetamol analysis. Furthermore, from a panel of human saliva samples, inter-individual variability was found to be <10% using this approach. This technique represents a promising tool for rapid and accurate emergency diagnosis.

We created novel Pd/CB-Ni@rGO nanomaterials for glucose detection. The as-synthesized nanomaterials were dropped on the electrode surface using the drop casting technique. The prepared electrode was then attached to a paper-based device containing the sample zone and the reaction zone, enabling plasma isolation and an enzymatic reaction for glucose detection in whole blood. The nanomaterials and surfaces of electrodes were characterized by FTIR, TEM, and SEM. The proposed approach is a disposable glucose detection method that is unaffected by protein fouling on the electrode, and it requires only one drop of human blood. Therefore, there is no need for extensive sample preparation, and there is less sample consumption. Under optimal conditions, Pd/CB-Ni@rGO can accurately measure blood glucose levels with a linear range of 7 to 7140 μM ( R 2 = 0.9986) and a low detection limit of 0.82 μM. Besides, the developed sensor shows excellent anti-interference capacity, stability, and satisfactory reproducibility and repeatability. Importantly, Pd/CB-Ni@rGO was successfully applied for glucose in whole blood from 4 volunteers, with results that correlated well with those obtained using an Accucheck glucometer at a 95% confidence level. Given its low cost, high accuracy, and ease of use, the blood glucose sensor holds significant potential for clinical use and broadens the area of future noninvasive sensor development.

Illicit drug trafficking and abuse is a significant public safety and health concern. Color tests are commonly used for drug screening, but their poor specificity results in false positives. This study demonstrates the combination of drug residue collection using pressure-sensitive adhesive paper, on-paper color testing, and post-reaction analysis by paper spray mass spectrometry (PS-MS) on both portable and benchtop ion trap MS. All steps, including residue collection, color testing, and paper spray analysis, were performed on the same piece of paper. Three common color tests were investigated: the cobalt thiocyanate test for cocaine, the Simon test for methamphetamine, and the Marquis test for phenethylamine stimulants and opiates. The detection threshold for color tests ranged from 1.25 to 10 μg on paper. Drug residues were successfully confirmed by paper spray MS at the color test threshold in all cases, except for heroin after reaction with the Marquis reagent, when using the portable MS. In this case, the MS detection threshold was 4-fold higher than the color test threshold. The stability of the color test products was assessed through a time study. Drug residues could be detected by MS at least 24 hours after reaction. A series of realistic samples, including false positives, were analyzed to demonstrate the technique's utility in real-world scenarios. Overall, combining color tests with PS-MS offers a rapid, low-cost method for the collection and analysis of illicit drugs.

This work reports the development of a rapid, simple and inexpensive colorimetric paper-based assay for the detection of the severe acute respiratory symptom coronavirus 2 (SARS-CoV-2) humanized antibody. The paper device was prepared with lamination for easy sample handling and coated with the recombinant SARS-CoV-2 nucleocapsid antigen. This assay employed a colorimetric reaction, which is followed by horseradish peroxidase (HRP) conjugated detecting antibody in the presence of the 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The colorimetric readout was evaluated and quantified for specificity and sensitivity. The characterization of this assay includes determining the linear regression curve, the limit of detection (LOD), the repeatability, and testing complex biological samples. We found that the LOD of the assay was 9.00 ng μL-1 (0.112 IU mL-1). The relative standard deviation was approximately 10% for a sample number of n = 3. We believe that our proof-of-concept assay has the potential to be developed for clinical screening of the SARS-CoV-2 humanized antibody as a tool to confirm infected active cases or to confirm SARS-CoV-2 immune cases during the process of vaccine development.

We report for the first time a highly sensitive and rapid quantitative method for the detection of Salmonella Typhimurium (S. Typhimurium) using a conductive immunosensor on a paper-based device (PAD). S. Typhimurium monoclonal antibodies (MA) were first immobilized on a paper-based device and then captured by S. Typhimurium. After an immunoreaction on the device, the polyclonal antibody-colloidal gold conjugate (PA-AuNPs) was dropped to bind with S. Typhimurium. After a complete sandwich reaction, a dark red color appeared on the paper-based device, which can be observed by the naked eye for a rapid screening test. The electrical conductivity of PA-AuNPs between the screen-printed electrodes on the paper-based device was also measured for an accurate quantitative analysis. The electrical conductivity correlated well with the concentration of S. Typhimurium, which was controlled by the amount of S. Typhimurium attached to the polyclonal antibody-colloidal gold conjugate. The device showed a linear correlation for the concentration of the S. Typhimurium in the range of 10-10 8 CFU mL -1 in a logarithmic plot, with an R 2 value of 0.9882 and a limit of detection (LOD) as low as 10 CFU mL -1 . This simple, highly sensitive, and rapid method for the S. Typhimurium detection was successfully performed within 30 min, and it can be developed into small portable measuring devices in order to facilitate preliminary screening tests.

Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum . The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution ( eluted disk ) as well as direct placement of the disk in the LAMP reaction ( in situ disk ). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.

COVID-19 has caused global health problems, and so rapid diagnosis is crucial to slow spread of the disease. Herein, a novel lab-on-paper screening method for SARS-CoV-2 Omicron BA.2 variant was developed using a gold nanoparticle-based colorimetric biosensor along with sensitive detection of SARS-CoV-2 antigen using laser desorption ionization-mass spectrometry (LDI-MS). As a result of antigen-antibody interaction, in the presence of SARS-CoV-2 antigen the gold nanoparticles undergo aggregation and change color from red to light purple, allowing for rapid determination of SARS-CoV-2 antigen with the naked eye. Furthermore, the lab-on-paper method can be directly applied as a substrate for sensitive quantitation of SARS-CoV-2 antigen in saliva using LDI-MS without the use of a conventional organic matrix and sample preparation. LDI-MS offers early diagnosis with high sensitivity, rapidity without sample preparation and lower cost per test compared with reverse transcriptase-PCR, which is crucial for preventing mortality in patients with underlying conditions. This method showed linearity over 0.01-1 μg mL -1 covering the cut-off value of 0.048 μg mL -1 for COVID-19 detection in human saliva. Moreover, a colorimetric sensor for urea was also fabricated in-parallel, for prediction of COVID-19 severity in patients with chronic kidney disease. The color change upon increasing urea concentration directly reflected kidney damage, which is related to increasing risk of mortality among patients with COVID-19. Hence, this platform might be a potential device for non-invasive diagnosis of SARS-CoV-2 Omicron BA.2 variant, which is the variant of most concern because it is transmitted more rapidly than the original SARS-CoV-2 virus and the Delta variant.

Cellular viability measurements quantify decreased proliferation or increased cytotoxicity caused by drug candidates or potential environmental toxins. Direct viability measures count each cell to provide an accurate readout. This approach can prove analytically challenging and time-consuming when cells are maintained in 3D structures akin to tissues or solid tumors. While less labor-intensive, indirect viability measures can be less accurate due to the heterogeneous structural and chemical microenvironment that arises when cells are maintained in tissue-like architectures and in contact with extracellular matrices. Here we determine the analytical figures of merit of five indirect viability assays in the paper-based cell culture platform we continue to develop in our laboratory: calcein-AM staining, the CellTiter-Glo assay, imaging fluorescent protein expression, propidium iodide staining, and the resazurin assay. We also determined the compatibility of each indirect assay with hypoxic conditions, intra-experimental repeatability, inter-experimental reproducibility, and ability to predict a potency value for a known antineoplastic drug. Our results show that each assay has benefits and drawbacks to consider when choosing the appropriate readout to answer a particular research question. We also highlight that only one indirect readout is unaffected by hypoxia, a commonly overlooked variable in cell culture that likely yields inaccurate viability measures.

An increasing number of patients are living with Alzheimer's disease (AD); thus, the need for a method to detect AD early and sensitively has become urgent, and the demand for an intelligent analytical platform is growing year by year. Abnormal levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to be indicative of AD. In this work, a novel conjugated polythiophene (CP) compound was successfully combined with CdTe quantum dots (QDs) to improve their selectivity and sensitivity. The QDs successfully enabled the detection of low concentrations of AChE by turning on the fluorescence of the CdTe/CP via the interaction between CP and thiocholine produced by ATCh hydrolysis and aggregation induced emission enhancement (AIEE). Under optimal conditions, we reached a low detection limit of 0.14 U L -1 , which is 7.9 times lower than that of pristine QDs. More importantly, an efficient, inexpensive, and disposable paper-based platform, which allows the efficient visual detection of AChE activity via the color variation of CdTe/CP, was designed. Moreover, the accuracy of the method was demonstrated by conducting a recovery test in human serum, in which the recoveries reached 107% and 110%, proving that CdTe/CP has considerable potential to be used for analyzing real biological samples. The advantages of this method are its simplicity, fast detection capability, affordability, and the fact that it can be used for on-site detection of AChE activity. Furthermore, it has certain guiding significance for detecting AD.

In this paper, a paper-supported analytical device based on a sandwich immunoreaction and luminescence resonance energy transfer (LRET) was reported for the visual and quantitative determination of a cancer biomarker, in which upconversion nanoparticles (UCNPs) were located on the surface of the paper as energy donors and gold nanoparticles (AuNPs) were used as energy acceptors. Upon the recognition of the cancer biomarker by two rationally selected antibodies, the upconversion luminescence was quenched by the AuNPs in a biomarker concentration-dependent manner. As a model target, CEA was detected using this immunosensor, and a linear relationship within 0.5-30 ng mL-1 was obtained in buffer solution, with a detection limit of 0.21 ng mL-1. The immunosensor was also applicable in 20-fold diluted human serum with a linear range of 0.5-30 ng mL-1 and a detection limit of 0.36 ng mL-1. This technique also realized the qualitative judgment of the critical concentration of CEA in serum samples by the naked eye. This approach displays great application potential for point-of-care testing in clinical applications, as well as the potentiality to be extended to other kinds of disease-related biomolecules.

Escherichia coli O157:H7 is a severe foodborne pathogen that causes lots of life-threatening diseases. In the search for a rapid, sensitive, portable and low-cost method to detect this pathogen, we developed a wax-printed paper-based enzyme-linked immunosorbent assay (P-ELISA) based on microfluidic paper-based analytical devices (μPADs), with the whole operation time being less than 3 h and only needing 5 μl samples for detection. The limit of detection (LOD) of E. coli O157:H7 reached 104 CFU ml-1, which is an order of magnitude higher than that of conventional ELISA (C-ELISA). The LOD in artificially contaminated beef samples is 1 CFU per 25 g after enriching the culture for 8 h. This method is superior to the molecular biology method in detection sensitivity and superior to C-ELISA and the national standard method in detection time and cost. Thus, the established P-ELISA method has good sensitivity, specificity and repeatability. It can be suitable for point-of-care testing without expensive and bulky instruments and can also provide a platform for detecting other pathogens, especially in areas that lack advanced clinical equipment.

Cobalt(ii) phthalocyanine (CoPc) was suspended in aqueous medium and the colloidal system was used as catalyst for the electrochemical determination of hydrogen peroxide on paper-based electrodes modified with carbon nanomaterials. H 2 O 2 was oxidised at 0.275 V vs. Ag pseudoreference electrode. This system was adapted to develop a glucose sensor with glucose oxidase immobilized on the cellulose electrode. CoPc suspended nanoparticles acted as nanoenzyme mimicking peroxidase activity and were combined with different carbon nanomaterials to form hybrids with optimised catalytic performance. GO-CoPc paper-based electrodes yielded the best results with a linear range of ∼12 μM to 49 mM for H 2 O 2 and 0.1 mM to 1 mM for glucose. Glucose was determined in physiological serum and juice samples with recoveries of 93.3 and 94.2% respectively. CoPc could replace HRP for the catalytic sensing of H 2 O 2 , without the need to be dissolved. This material can be used in situ in a simple protocol with other nanomaterials for electrode modification. The sensor described has the advantage of easy preparation, using the catalyst in colloidal form, long term stability, and versatility to be adapted to other low cost and disposable enzymatic systems.

Binding protein scaffolds, such as rcSso7d, have been investigated for use in diagnostic tests; however, the functional performance of rcSso7d has not yet been studied in comparison to antibodies. Here, we assessed the analyte-binding capabilities of rcSso7d and antibodies on cellulose with samples in buffer and 100% human serum.

Nearly 7% of the world's population live with a hemoglobin variant. Hemoglobins S, C, and E are the most common and significant hemoglobin variants worldwide. Sickle cell disease, caused by hemoglobin S, is highly prevalent in sub-Saharan Africa and in tribal populations of Central India. Hemoglobin C is common in West Africa, and hemoglobin E is common in Southeast Asia. Screening for significant hemoglobin disorders is not currently feasible in many low-income countries with the high disease burden. Lack of early diagnosis leads to preventable high morbidity and mortality in children born with hemoglobin variants in low-resource settings. Here, we describe HemeChip, the first miniaturized, paper-based, microchip electrophoresis platform for identifying the most common hemoglobin variants easily and affordably at the point-of-care in low-resource settings. HemeChip test works with a drop of blood. HemeChip system guides the user step-by-step through the test procedure with animated on-screen instructions. Hemoglobin identification and quantification is automatically performed, and hemoglobin types and percentages are displayed in an easily understandable, objective way. We show the feasibility and high accuracy of HemeChip via testing 768 subjects by clinical sites in the United States, Central India, sub-Saharan Africa, and Southeast Asia. Validation studies include hemoglobin E testing in Bangkok, Thailand, and hemoglobin S testing in Chhattisgarh, India, and in Kano, Nigeria, where the sickle cell disease burden is the highest in the world. Tests were performed by local users, including healthcare workers and clinical laboratory personnel. Study design, methods, and results are presented according to the Standards for Reporting Diagnostic Accuracy (STARD). HemeChip correctly identified all subjects with hemoglobin S, C, and E variants with 100% sensitivity, and displayed an overall diagnostic accuracy of 98.4% in comparison to reference standard methods. HemeChip is a versatile, mass-producible microchip electrophoresis platform that addresses a major unmet need of decentralized hemoglobin analysis in resource-limited settings.

Rapid analysis of metabolites in biofluids is of great importance for disease diagnosis or new-born disease screening. Herein, we introduce an agarose hydrogel conditioning method to enhance the performance of paper spray ionization mass spectrometry. With facile and fast hydrogel conditioning, the signal intensity of therapeutic drugs spiked in urine was 5 to 15 fold higher than that in direct paper spray ionization mass spectrometry analysis. Consequently, the sensitivity of metabolites in urine was improved via hydrogel conditioning, resulting in 9 to 15 fold decrease in the possibility of detection (POD) levels. These results show that agarose hydrogel conditioning coupled with paper spray ionization mass spectrometry could serve as a facile ionization method for ambient mass spectrometry, which might be useful in fast screening of metabolites and therapeutic drugs in raw biofluids.

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg μL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg μL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg μL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.

EVs/exosomes are considered as the next generation of biomarkers, including for liquid biopsies. Consequently, the quantification of EVs/exosomes is crucial for facilitating EV/exosome research and applications. Paper-based enzyme-linked immunosorbent assay (p-ELISA) is a portable diagnostic system with low cost that is simple and easy to use; however, it shows low sensitivity and linearity. In this study, we develop p-ELISA for targeting EVs/exosomes by using streptavidin agarose resin-based immobilization (SARBI). This method reduces assay preparation times, provides strong binding, and retains good sensitivity and linearity. The time required for the total assay, including preparation steps and surface immobilization, was shortened to ∼2 h. We evaluated SARBI p-ELISA systems with/without CD63 capture Ab and then with fetal bovine serum (FBS) and EVs/exosome-depleted fetal bovine serum (dFBS). The results provide evidence supporting the selective capture ability of SARBI p-ELISA. We obtain semiquantitative p-ELISA results using an exosome standard (ES) and human serum (HS), with R2 values of 0.95 and 0.92, respectively.

This communication describes the rational design of a transparent paper-based chemosensing platform for multi-target detection by wavelength-dependent absorbance/transmittance. The platform was successfully applied in the examination of bovine serum albumin (BSA) and cholesterol in serum with a low detection limit of 0.1 μM and 0.1 mM, respectively. With low cost and high sensitivity, the paper-based platform shows great promise for multiplexed bioassays.

An accurate quantification method with a wide linearity range is paramount for the development of low-cost, portable and point-of-care sensors. This work reports a new approach to analyze the colorimetric assays on paper-based sensors using the quantification from a light transmission method. Compared to the commonly-developed color intensity measurement on scanned digital images, a portable transmission densitometer is capable of directly quantifying the optical density of colorimetric results. The detection of heavy metals in an aqueous system, including Fe(ii), Cu(ii), and Ni(ii), was carried out to demonstrate the good performance and reliability of this method. Our measurements show that the linear quantification range spans from 0.5-500 mg L -1 for the assays of Cu(ii) and Fe(ii) and from 2-500 mg L -1 for Ni(ii) based on the reading of transmitted light through the assay spot. As a comparison, the linear range is restricted to 0.5-50 mg L -1 for the same assays when analysed by the common reflection method, suggesting a significant improvement in the accuracy and sensitivity of high analyte concentrations from the light transmission method. By expanding the linearity range, this method further streamlines the sampling procedure during analysis and will greatly advance the future development of paper-based analytical sensors.

In the emergency diagnosis of patients, acute myocardial infarction (AMI) is always time-consuming to diagnose, and the process requires multiple laboratory procedures, expensive equipment and skilled workers. Herein, we developed an easy-to-use, low-cost and portable fluorescent lateral flow immunoassay based on paper microfluidics for the point-of-care diagnostics of non-communicable diseases. The fluorescent lateral flow immunoassay can produce results in less than 10 minutes, and the limit of detection (LOD) is 0.019 ng ml -1 . The slope was linear from 0 to 100 ng ml -1 ; the equation is y = 0.0342e 2.1181x and R 2 = 0.9618, which are distinctive features that ensure maximum amplification of the signal and recording of quantitative values by an analyser. The detection sensitivity showed an exceptional increase to 0.01 ng ml -1 . Compared with conventional bioassay readers, our analyser shows some advantages to easily, clearly and effectively read data. The present point-of-care test for cardiac troponin I decreases the turnaround time and has a high coefficient of variation even at lower concentrations of troponin. So, the development of lateral flow assay-based point-of-care assays with higher analytical performance for real world samples can decrease the rule-out time for AMI in emergency departments and other fields.

Fibrinogen is a blood protein that is essential for clotting. It is converted into the polymer fibrin by the blood enzymes thrombin and factor XIIIa. Fibrinogen is one of the first proteins to be depleted in heavily bleeding patients. Patients with early hypofibrinogenemia need urgent fibrinogen replenishment to prevent the onset of haemorrhage and death. However, currently there is no rapid, sensitive, cheap and easy-to-use fibrinogen assay that can detect fibrinogen concentrations. In this study, we have developed a new paper-based diagnostic to quantify the fibrinogen concentration in blood at room temperature. This diagnostic is a 2-step process: first, plasma is added onto thrombin-treated paper strips where fibrinogen is converted to fibrin; then the strips are placed into an aqueous dye bath where elution occurs. The test operates by measuring the change in hydrophobicity, which increases with fibrinogen concentration under otherwise constant conditions. The diagnostic can precisely measure fibrinogen concentration within the range of 0-2 g L-1, which is ideal for the clinical diagnosis of hypofibrinogenemia. Furthermore, testing needs only 12 μL of plasma, 60 mU of thrombin and 7.5 minutes of testing. This diagnostic has the potential to revolutionise point of care testing and save many lives.

Sweat, as a sample that includes a lot of biochemical information, is good for non-invasive monitoring. In recent years, there have been an increasing number of studies on in situ monitoring of sweat. However, there are still some challenges for the continuous analysis of samples. As a hydrophilic, easy-to-process, environmentally friendly, inexpensive and easily accessible material, paper is an ideal substrate material for making in situ sweat analysis microfluidics. This review introduces the development of paper as a sweat analysis microfluidic substrate material, focusing on the advantages of the structural characteristics of paper, trench design and equipment integration applications to expand the design and research ideas for the development of in situ sweat detection technology.

In 2022, the outbreak of the monkeypox virus occurred in many non-endemic countries, and the World Health Organization (WHO) assessed that this outbreak was "atypical". The establishment of a rapid and effective assay that can be used for the early diagnosis of monkeypox virus infection is crucial for outbreak prevention and control. In this study, the monkeypox virus A29 protein and the homologous vaccinia virus A27 protein and cowpox virus 162 protein were expressed in Escherichia coli BL21 for screening. We synthesized the monkeypox virus A29 17-49 peptide as the immunogen and obtained 25 monoclonal antibodies (mAbs) against the A29 protein using mouse hybridoma techniques. Then an immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity and lowest limit of detection: 50 pg mL -1 for purified A29 and specificity tests showed that the strips did not cross-react with other orthopox viruses (vaccinia virus or cowpox virus) as well as common respiratory pathogens (SARS-CoV-2, influenza A and influenza B). Therefore, this method can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection.

Paper-based microfluidics is an emerging analysis tool used in various applications, especially in point-of-care (PoC) diagnostic applications, due to its advantages over other types of microfluidic devices in terms of simplicity in both production and operation, cost-effectiveness, rapid response time, low sample consumption, biocompatibility, and ease of disposal. Recently, various techniques have been developed and utilized for the fabrication of paper-based microfluidics, such as photolithography, micro-embossing, wax and PDMS printing, etc . In this study, we offer a fabrication methodology for a microfluidic paper-based immunosorbent assay (μPISA) platform and the detection of Hepatitis C Virus (HCV) was carried out to validate this platform. A laser ablation technique was utilized to form hydrophobic barriers easily and rapidly, which was the major advantage of the developed fabrication methodology. The characterization of the μPISA platform was performed in terms of micro-channel properties using bright-field (BF) microscopy, and surface properties using scanning electron microscopy (SEM). At the same time, sample volume and liquid handling capacity were analyzed quantitatively. Ablation speed ( S ) and laser power ( P ) were optimized, and it was shown that one combination (10P60S) provided minimal deviation in micro-channel dimensions and prevented deterioration of hydrophobic barriers. Also, the minimum hydrophobic barrier width, which prevents cross-barrier bleeding, was determined to be 255.92 ± 10.01 μm. Furthermore, colorimetric HCV NS3 detection was implemented to optimize and validate the μPISA platform. Here, HCV NS3 in both PBS and human blood plasma was successfully detected by the naked eye at concentrations as low as 1 ng mL -1 and 10 ng mL -1 , respectively. Moreover, the limit of detection (LoD) values for HCV NS3 were acquired as 0.796 ng mL -1 in PBS and 2.203 ng mL -1 in human blood plasma with a turnaround time of 90 min. In comparison with conventional ELISA, highly sensitive and rapid HCV NS3 detection was accomplished colorimetrically on the developed μPISA platform.

The fast and precise detection of potential allergen-specific immunoglobulin E (sIgE) is imperative for the diagnosis and appropriate treatment of allergic diseases. In this study, we have successfully fabricated a novel paper-based immunoassay device for the detection of sIgE in allergic diseases. We used Can f 1, one of the main dog allergens, as a model allergen to detect sIgE in human sera. To achieve excellent performance, the experimental parameters were optimized. Further, we extended this device for potential applications in the clinical diagnosis of allergic diseases: worthwhile clinical performance in the detection of allergens was achieved as compared to that achieved by commercial enzyme-linked immunosorbent assay (ELISA) kit. Therefore, it was proven that this strategy has the advantages of high-throughput, rapid, sensitive, and highly accurate detection of trace amounts of sIgEs. Furthermore, by simply changing the antigen and antibody, this device could be used for the high-throughput detection of other allergens, so as to achieve multiallergen detection and appropriate desensitization therapy, thereby making it promising in the determination of allergic diseases in clinics.

Managing food contamination from bacteria has been an ongoing issue in the public health and industrial fields. Enzymatic substrates possessing optical properties, e.g. fluorescence or color manifestation, are widely exploited in pathogenic/non-pathogenic bacteria culture methods. Recently, various chromogenic substrates have been utilized in the development of point-of-care diagnostic tools. Herein, four types of chromogenic substrates were exploited to develop paper-based sensors for major foodborne pathogens. We designed a compact sized three-dimensional paper device with a simple user interface. By inserting functional layers in the middle of multilayers, pre-lysis and pH regulation steps were excluded and the analysis time was subsequently reduced, while only one sample droplet was needed for the whole analysis process. After the enzymatic reactions had proceeded, target-specific colors appeared. When it was combined with enrichment, 101 cfu mL-1 of pathogens were successfully detected in 4-8 hours, while those in milk samples were readily sensed in 12 hours. The proposed bacteria sensor exhibited great advantages of low cost, portability and simple operation, while showing a respectable limit-of-detection as low as 101 cfu mL-1 and below. Significantly, we emphasize that it takes fewer steps than existing methods and provides a reduced analysis time owing to the layer functionalization.

A paper-based platform was investigated in which the selective detection of oligonucleotide targets by hybridization was accomplished via the enhancement of fluorescence emission from intrinsically labeled DNA probes that were immobilized on the surface of quantum dots (QDs). Multiple copies of a derivative of thiazole orange, an intercalating dye known to form non-emissive dimers, were conjugated to single-stranded oligonucleotide probes. Dimerization resulted in the formation of H-aggregates where excitonic interactions led to the suppression of fluorescence. The hybridization of the oligonucleotide probe with a complementary target resulted in the enhancement of fluorescence emission as the dimers dissociated and the dyes preferentially intercalated with the duplex. The detection of oligonucleotide targets using this configuration eliminated the need for labeling the target strands, and fluorescence intensity was proportional to the extent of hybridization. In addition, the dye molecules were excited using Foerster Resonance Energy Transfer (FRET) from QD donors, which resulted in improved selectivity and allowed for ratiometric detection. A solution-phase hybridization assay based on similar operational principles has been previously reported, and this new work investigated the advantages offered for this transduction scheme using paper-based solid-phase substrates. QD-probe conjugates were immobilized in sufficient density on the paper matrix to provide for multiple-donor-multiple-acceptor interactions that resulted in a 20-fold enhancement of acceptor emission compared to the solution-based assay, providing a limit of detection of 0.1 pmol. The paper-based assay provided for the reduction of the time needed for sample preparation and data acquisition, demonstrated that transduction was possible in a complex matrix (goat serum) without compromising on the performance observed in buffer solution, and that oligonucleotides generated from standard PCR amplification could be detected.

Rapid detection of pesticides in fruits is an ongoing challenge. The objective of the present study was to develop novel fluorescent microfluidic paper chips for specific recognition and sensitive detection of the pesticide 2,4-D through the electron-transfer-induced fluorescence quenching mechanism. CdTe quantum dots (QDs) were deposited onto cellulose paper (base material) to yield imprinted paper chips (paper@QDs@MIPs). This method allows the transferability of the molecularly imprinted fluorescence sensor from the liquid phase to the solid phase (paper base) for rapid and portable analysis. The resultant imprinted paper chips were effectively characterized, and they exhibited ideal ordered spatial network structure, chemical stability, and fluorescence property. The paper@QDs@MIPs showed that 2,4-D binding significantly reduced the fluorescence intensity within less than 18 min, and it achieved satisfactory linearity in the range of 0.83-100 μM and high detectability of 90 nM. The recognition specificity for 2,4-D relative to its analogues was shown, and the imprinting factor was 2.13. In addition, the recoveries of the spiked bean sprouts at three concentration levels ranged within 94.2-107.0%, with a relative standard deviation of less than 5.9%. Collectively, the device provided an effective platform for rapid recognition, convenience, and detection of trace food pollutants in complex matrices, thereby ensuring food safety and further promoting surface imprinting studies.

Cellular movement is essential in the formation and maintenance of healthy tissues as well as in disease progression such as tumor metastasis. In this work, we describe a paper-based Transwell assay capable of quantifying cellular invasion through an extracellular matrix. The paper-based Transwell assays generate similar datasets, with equivalent reproducibility, to commercially available Transwell assays. With different culture configurations, we quantify invasion: upon addition of an exogenous factor or in the presence of medium obtained from other cell types, in an indirect or direct co-culture format whose medium composition is dynamically changing, and in a single-zone or parallel (96-zone) format.

Recently, paper has gained traction in the biotechnology research field due to its ability to be a substrate for 3D cell culture. In this work, we demonstrate the application of paper-based 3D cell culture for rapid and easy screening of the effect of natural compounds on melanin production. Whatman No. 1 filter paper was used as the substrate for B16F10 melanoma cell culture. The use of paper is beneficial for supporting the 3D structure of cells, which makes the result more reliable due to the similarity to in vivo conditions. Furthermore, paper is beneficial for melanin observation due to melanin's black color, which is easily in situ visualized after it is cultured on white paper. Matrigel was used to encapsulate cells before being pipetted onto the paper to prevent the passing of cells through paper pores. The intensity of melanin can then be observed with the naked eye and analyzed by scanning the paper. The analysis process took only 20 minutes, which is faster than that of the conventional absorbance spectroscopy, owing to the elimination of centrifugation, melanin solubilization, and the absorbance measurement step. The color intensity on the paper showed a direct proportion with increased α-MSH concentrations, confirming that the color on the paper was melanin. The 3D structure of cells was confirmed by using a scanning electron microscope. To demonstrate the application of the paper-based scaffold, paper-based 3D cell culture was used for screening the effects of Kojic acid and Arbutin on melanin production, which showed increased anti-melanogenesis effects with increased concentrations of natural compounds. High cell viability was observed over 120 hours. In conclusion, the developed paper-based scaffold can be used for screening the effect of natural compounds on melanin production, as a rapid and simple method with low cost.

A novel paper-based analytical device (PAD) was fabricated and developed for the simple and rapid determination of the albumin to creatinine ratio (ACR) in urine samples. The detection was based on a colorimetric reaction using bromocresol green (BG) in a phosphate buffer (PB) at pH 4 for the determination of albumin (AL) and creatinine (CR). BG changes color from greenish-yellow to bluish-green in the presence of AL and/or CR. Picric acid (PA) in 0.25 M NaOH was used to detect CR, and PA changes color from yellow to orange. Under the optimal conditions, the working range was 10 to 350 mg dL-1 with a detection limit of 7.1 and 5.4 mg dL-1 for AL + CR and CR detection, respectively. The repeatability was evaluated, and the %RSD value was less than 8.23 (n = 10). The ACR was obtained by calculating the AL and CR colorimetric results. Finally, the proposed devices were applied to the determination of AL, CR, and ACR in urine samples. The results obtained by the developed PADs were in good agreement with the standard method and demonstrated the method could reliably measure AL, CR, and ACR. The proposed method provides a low-cost, simple, sensitive, and promising tool for diagnostic identification assay for chronic kidney disease (CKD).

In this work, a versatile point-of-care assay platform based on a microfluidic paper-based analytic device (μPAD) was developed for the simultaneous detection of multiple targets. The μPAD with a central zone and six test zones is fabricated by a simple and inexpensive wax printing method. A flower-like hybrid nanocomplex synthesized with specific dual enzymes and Cu3(PO4)2 inorganic nanocrystals is spotted in the test zones on the μPAD, followed by the introduction of assay targets. Using dual-enzyme inorganic hybrid nanoflowers in the μPAD as nanobiocatalysts, which preserve the activity and enhance the stability of the enzymes, based on the H2O2-mediated catalytic oxidizing chromogenic reaction produced by glucose/uric acid, the developed multiplex paper-based nanobiocatalytic system is demonstrated to enable simultaneous and sensitive detection of glucose and uric acid with a detection limit of 60 and 25 μM, respectively. More importantly, it has been successfully used for detecting glucose and uric acid levels in human whole blood samples. The developed multiplex paper-based nanobiocatalytic system features very easy fabrication and operation, low cost, and high sensitivity and has promising prospects for a clinical multianalyte point-of-care test.

Sodium thiocyanate (NaSCN) is a naturally antibacterial component in milk, but excessive consumption of thiocyanate may cause potential risks to human health. Currently available methods for the detection of thiocyanate have some disadvantages such as poor sensitivity and high price. Here, we report a robust and cost-effective method to detect NaSCN based on paper substrates deposited with in situ reduced Ag nanoparticles by surface-enhanced Raman spectroscopy (SERS). Densely packed multilayer AgNPs provide uniform narrow nanogaps, which exponentially enhance the Raman signals. Moreover, these homogeneous narrow hotspots ensure that this method has high sensitivity (the limit of detection is 10 -12 mol L -1 ), a wide linear range (from 10 -9 mol L -1 to 10 -4 mol L -1 ), and remarkable reproducibility (the coefficient of variation within a SERS sensor is 6.5%). Spiked milk samples were detected and the recovery rates of NaSCN were in the range of 95.1%-108.0%. This paper-based SERS sensor offers great potential for sensitive NaSCN detection and milk analysis.

Herein, a unique electrochemiluminescence (ECL) sensor combined with a paper electrode was proposed for the detection of phenylalanine (L-Phe) in blood samples. The L-Phe detection was performed by converting L-Phe into ammonia using phenylalanine ammonia-lyase (PAL) enzyme and the ECL signal of Ru(bpy) 3 was produced in combination with ammonia as a co-reactant. In this report, we used AuNP decorated paper electrodes to obtain the ECL signal of Ru-(bpy) 2+ was produced in combination with ammonia as a co-reactant. In this report, we used AuNP decorated paper electrodes to obtain the ECL signal of Ru-(bpy) 3 2+ in the presence of NH 3 . The produced ammonia molecules were carried to the working electrode and their contact with the sample was cut off. Since there was no contact between the sample matrix and the electrode, the developed method achieved excellent selectivity. According to experimental data, a linear increase of the ECL signals with the logarithms of varying L-Phe concentrations between 1.5 × 10 -2 mM. The proposed method was efficiently applied for L-Phe detection in reference blood samples and the average recovery was calculated as 96.8%. Furthermore, the HPLC-UV method as a comparison verified that the recovery values provided by the proposed method were acceptable with a similarity of 95.1% for L-Phe detection in a reference blood sample. The results showed that the developed ECL sensor combined with the paper electrode can be considered as a promising unique and powerful technique for a point-of-care diagnosis of PKU.R 2 ) of 0.9898 and a limit of detection (LOD) of 8.4 × 10 -3 mM. The proposed method was efficiently applied for L-Phe detection in reference blood samples and the average recovery was calculated as 96.8%. Furthermore, the HPLC-UV method as a comparison verified that the recovery values provided by the proposed method were acceptable with a similarity of 95.1% for L-Phe detection in a reference blood sample. The results showed that the developed ECL sensor combined with the paper electrode can be considered as a promising unique and powerful technique for a point-of-care diagnosis of PKU.

Amino acids are closely related to human health, and their rapid determination is important for the rapid diagnosis, timely treatment, and assessment of serious diseases. In this work, we propose a novel paper-based sample-processing device combined with isotope-dilution MS for the fast analysis of 11 amino acids from blood samples. By using an isoelectric focusing electrokinetic separation method, without the aid of carrier ampholytes and the addition of inhibitors, this approach uses only the characteristic of the isoelectric point of the target amino acids to achieve separation and purification from other coexisting components in the medium; it can meet the requirements for mass spectrometry detection. Driven by a DC voltage, a stable and sharp pH gradient (pH 3-10.5 over 5 mm) can be established in a glass-fiber paper-based fluidic channel with a MS-friendly electrolyte. Amphoteric species can be well separated from the complex blood matrix and concentrated into a narrow band in the channel within 2 min, which is 20 times faster than a commercial kit method. The method can be applied to both liquid and dry spot samples, and the cleaned sample band can be simply dissolved for direct IDMS detection in ESI MRM mode. This method is a promising strategy for the rapid MS-based detection of amino acids from serum without pre-separation via liquid chromatography.

Paper-based microfluidic solution sampling is a viable option for potentiometric sensors to be used for the determination of analytes in samples with high solid-to-liquid ratios. Unfortunately, heavy metal sensitive electrodes cannot be easily integrated with paper-based solution sampling as heavy metals have strong physicochemical adsorption affinity towards paper substrates. In this work, paper substrates were modified with an ion-selective membrane (ISM) cocktail (used for the preparation of Pb 2+ -ion-selective electrodes (ISEs)) and coupled with model heavy metal Pb 2+ -ISEs. It was found that the super-Nernstian response of Pb 2+ -ISEs was eliminated when 10 to 50 mg ml -1 of the ISM cocktail was used for the modification of paper substrates. The modification of the paper substrates by Pb 2+ -ISM allowed the elimination of adsorption sites. In addition, it resulted in an improvement of sensor performance in terms of their detection limits to be similar to those for conditioned electrodes in standard beaker-based measurements. It is believed that the elimination of super-Nernstian response of the electrodes and improving the potentiometric responses and detection limits of ISEs were attributed to the compatibility improvement of the paper substrates and Pb 2+ -ISEs to the same type of ISM.

The achievement of high-throughput separation and high-sensitivity detection of complex samples has been one of the most challenging issues in the field of analytical science. The application of a single technology alone could not satisfy the above requirements. The combination of technologies with the capability of high-efficiency separation and high-sensitivity structural-recognition is highly desired to meet the technical requirements. Herein, an automatic high-performance liquid chromatography (HPLC)-surface enhanced Raman spectroscopy (SERS) hyphenated system using paper substrates as the "interface" was constructed to achieve efficient separation and real-time detection. A homogeneous Au nanoparticle was printed on the hydrophobic filter paper with the inkjet technology. The prepared substrates served as a linkage for the continuous realization of HPLC and SERS functions. The complex system was separated by HPLC, and the effluents were loaded onto automatically and continuously replaceable paper substrates for real time SERS measurements. The continuous rapid separation and real-time detection of various two-component mixtures were achieved with the separation efficiency and detection sensitivity of each technology. The results demonstrated that the HPLC-SERS hyphenated system exhibited the complementary capability of the on-line separation and continuous structural identification of illegal additives in real samples. The detection sensitivity was increased by an order of magnitude to reach 10 -5 mol dm -3 , and the efficiency and accuracy for the separation and identification on the multi-components samples were higher than those of the individual HPLC or SERS technology. It is believed that the continuous paper substrate-based HPLC-SERS hyphenated system would be developed as a promising technique for the separation and identification of multi-components mixtures with high throughput.

Bioelectrochemical technologies have attracted significant scientific interest because the effective bacterial electron exchange with external electrodes can provide a sustainable solution that joins environmental remediation and energy recovery. Multispecies electroactive bacterial biofilms are catalysts that will drive the operation of bioelectrochemical devices. Unfortunately, there is a lack of understanding of key mechanisms determining their electron-generating capabilities and syntrophic relations within microbial communities in biofilms. This is because there are no universally standardized models for simple, rapid, reliable, and cost-effective fabrication and characterization of electroactive multispecies biofilms. The heterogeneous and long-term nature of biofilm formation has hampered the development of those models. This work develops novel biofabrication and analysis platforms by creating innovative, paper-based 3-D systems that accurately recapitulate the structure, function, and physiology of living multispecies biofilms. Multiple layers of paper containing bacterial cells were stacked to simulate different layered 3-D biofilm models with defined cellular compositions and microenvironments. Overall bacterial electrogenic capabilities through the biofilm structures were characterized by thoroughly monitoring collective electron flows through different external resistors. Changes in the type of species and order of stacking created biofilm modeling which allowed for the study of their electrogenic performance via variation in electron flow rate output. Furthermore, multi-laminate structures allowed for straightforward de-stacking and layer-by-layer separation for analyses of pH distribution and cellular viability. Our multi-laminate structures provide a new strategy for (i) controlling the biofilm geometry of 3-D bacterial cultures, (ii) monitoring the microbial electoral properties, and (iii) constructing an artificial biofilm layer by layer.