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The existence of pesticide residues in the hydrosphere, biosphere, and anthroposphere can cause acute or chronic diseases and deteriorate the environment. Therefore, efficient detection of pesticide residues is of great significance to prevent food poisoning, control food pollution, and protect human lives by recognizing their distribution and concentration. Herein, a novel smartphone-coupled three-layered paper-based microfluidic chip is proposed as a facile platform to detect the pesticides. The stereoscopic capillary-driven fluid transport is enabled by the three-layered microfluidic chip configuration. The detection mechanism is based on the enzyme inhibition reaction and the chromatic reaction. The detection results are obtained by a smartphone and figured out by colorimetric quantitative analysis. Taking advantages of the above merits, we demonstrate the utilization of this smartphone-coupled three-layered paper-based microfluidic chip for the effective analysis of typical pesticides (profenofo and methomyl). The linear ranges of profenofo and methomyl are 0.27-2.1 μmol L

Paper has been widely employed as cheap material for the development of a great number of sensors such as pregnancy tests, strips to measure blood sugar, and COVID-19 rapid tests. The need for new low-cost analytical devices is growing, and consequently the use of these platforms will be extended to different assays, both for the final consumer and within laboratories. This work describes a paper-based electrochemical sensing platform that uses a paper disc conveniently modified with recognition molecules and a screen-printed carbon electrode (SPCE) to achieve the detection of gluten in a deep eutectic solvent (DES). This is the first method coupling a paper biosensor based on aptamers and antibodies with the DES ethaline. Ethaline proved to be an excellent extraction medium allowing the determination of very low gluten concentrations. The biosensor is appropriate for the determination of gluten with a limit of detection (LOD) of 0.2 mg L−1 of sample; it can detect gluten extracted in DES with a dynamic range between 0.2 and 20 mg L−1 and an intra-assay coefficient of 10.69%. This approach can be of great interest for highly gluten-sensitive people, who suffer from ingestion of gluten quantities well below the legal limit, which is 20 parts per million in foods labeled gluten-free and for which highly sensitive devices are essential. Graphical abstract

Iron is an important micronutrient involved in several mechanisms in the human body and can be an important biomarker. In this work, a simple and disposable microfluidic paper-based analytical device (µPAD) was developed for the quantification of iron in urine samples. The detection was based on the colorimetric reaction between iron(II) and bathophenanthroline and the reduction of iron(III) to iron(II) with hydroxylamine. The developed µPAD enabled iron determination in the range 0.07–1.2 mg/L, with a limit of detection of 20 µg/L and a limit of quantification of 65 µg/L, thus suitable for the expected values in human urine. Additionally, targeting urine samples, the potential interference of the samples color was overcome by incorporating a sample blank assessment for absorbance subtraction. Stability studies revealed that the device was stable for 15 days prior to usage and that the formed colored product was stable for scanning up to 3 h. The accuracy of the developed device was established by analyzing urine samples (#26) with the developed µPAD and with the atomic absorption spectrometry method; the relative deviation between the two sets of results was below 9.5%. Graphical abstract: [Figure not available: see fulltext.]

High per- and polyfluorinated alkyl substance (PFAS) concentrations have been detected in agricultural soils in Southwest Germany. Discharges of PFAS-contaminated paper sludge and compost are suspected to be the cause of the contamination. Perfluorinated carboxylic acids (PFCAs) have been detected also in groundwater, drinking water, and plants in this area. Recently, previously unknown compounds have been identified by high-resolution mass spectrometry (HRMS). Major contaminants were polyfluorinated dialkylated phosphate esters (diPAPs) and N-ethyl perfluorooctane sulfonamide ethanol–based phosphate diester (diSAmPAP). In this study, HRMS screening for PFAS was applied to 14 soil samples from the contaminated area and 14 impregnated paper samples which were from a similar period than the contamination. The paper samples were characterized by diPAPs (from 4:2/6:2 to 12:2/12:2), fluorotelomer mercapto alkyl phosphates (FTMAPs; 6:2/6:2 to 10:2/10:2), and diSAmPAP. In soil samples, diPAPs and their transformation products (TPs) were the major contaminants, but also FTMAPs, diSAmPAP, and their TPs occurred. The distribution patterns of the carbon chain lengths of the precursor PFAS in soil samples were shown to resemble those in paper samples. This supports the hypothesis that paper sludge is a major source of contamination. The presence of major degradation products like PFCAs, FTSAs, or PFOS and their distribution of carbon chain lengths indicate the activity of biotic or abiotic degradation processes and selective leaching processes from the upper soil horizons. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03463-9.

Urinary tract infections (UTIs) make up a significant proportion of the global burden of disease in vulnerable groups and tend to substantially impair the quality of life of those affected, making timely detection of UTIs a priority for public health. However, economic and societal barriers drastically reduce accessibility of traditional lab-based testing methods for critical patient groups in low-resource areas, negatively affecting their overall healthcare outcomes. As a result, cellulose-based materials such as paper and thread have garnered significant interest among researchers as substrates for so-called frugal analytical devices which leverage the material’s portability and adaptability for facile and reproducible diagnoses of UTIs. Although the field may be only in its infancy, strategies aimed at commercial penetration can appreciably increase access to more healthcare options for at-risk people. In this review, we catalogue recent advances in devices that use cellulose-based materials as the primary housing or medium for UTI detection and chart out trends in the field. We also explore different modalities employed for detection, with particular emphasis on their ability to be ported onto discreet casings such as sanitary products. Graphical abstract

Graphical abstract Enzyme-linked immunosorbent assay (ELISA) is widely used for the detection of disease biomarkers. However, it utilizes time-consuming procedures and expensive instruments, making it infeasible for point-of-care (POC) analysis especially in resource-limited settings. In this work, a multicolorimetric ELISA biosensor integrated on a paper/polymer hybrid microfluidic device was developed for rapid visual detection of disease biomarkers at point of care, without using costly equipment. This multicolormetric ELISA platform was built on multiple distinct color variants resulted from the catalytic oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) and the etching of gold nanorods (AuNRs). The vivid color changes could be easily distinguished by the naked eye, and their red mean values allowed quantitative biomarker detection, without using any sophisticated instruments. When this multicolorimetric ELISA was integrated on a paper/polymer hybrid analytical device, it not only provided integrated processing and high portability but also enabled fast assays in about 50 min due to the unique advantages of paper/polymer hybrid devices. The limit of detection of 9.1 ng/μL of the hepatitis C virus core antigen, a biomarker for hepatitis C, was achieved using this multicolorimetric ELISA platform. This multicolor ELISA analytical device provides a new versatile, user-friendly, affordable, and portable immunosensing platform with high potential for on-site detections of various viruses, proteins, and biomarkers for low-resource settings such as at home, public venues, rural areas, and developing nations. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03359-8.

Graphical abstract During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP—KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3—to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3–25 nM) exhibited the lowest limits of detection (ca. μg mL−1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual’s immune response can impact the interpretation of assay results. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03317-4.

Microfluidic paper-based analytical devices (μPADs) have developed rapidly in recent years, because of their advantages, such as small sample volume, rapid detection rates, low cost, and portability. Due to these characteristics, they can be used for in vitro diagnostics in the laboratory, or in the field, for a variety of applications, including food evaluation, disease screening, environmental monitoring, and drug testing. This review will present various detection methods employed by μPADs and their respective applications for the detection of target analytes. These include colorimetry, electrochemistry, chemiluminescence (CL), electrochemiluminescence (ECL), and fluorescence-based methodologies. At the same time, the choice of labeling material and the design of microfluidic channels are also important for detection results. The construction of novel nanocomponents and different smart structures of paper-based devices have improved the performance of μPADs and we will also highlight some of these in this manuscript. Additionally, some key challenges and future prospects for the use of μPADs are briefly discussed.

Process-monitoring laboratories in the pulp and paper industry generally use a combination of wet chemical analyses and physical measurements to certify the fitness of a production pulp for a specific end-use. These laboratory tests require time and the effort of trained personnel, limiting their utility for real-time process control. Here we show that Raman probes of unrefined cellulosic pulps, well-suited to the online measurement of in-process materials, can predict the quality attributes of manufactured papers. The accuracy of prediction improves when the covariance is modelled in a reduced measurement space selected by a data-driven, feature-selection technique referred to as a Template Oriented Genetic Algorithm (TOGA). TOGA, combined with discrete wavelet transform (DWT), isolates functional-group features that correlate best with mechanical properties paper derived from refined pulp. Paper makers refine market pulps to build sheet strength using a beating process that decreases freeness as it increases fibre-fibre bonding. Methods demonstrated here predict manufactured sheet properties obtainable after any specified degree of refining from the Raman spectrum of an unrefined pulp. This analysis capacity will enable both vendors of market pulp and makers of sheet paper to specify in advance the amount of beating required to produce a desired product, thereby saving cost and conserving resources.

Pathogen detection is crucial for human, animal, and environmental health; crop protection; and biosafety. Current culture-based methods have long turnaround times and lack sensitivity. Nucleic acid amplification tests offer high specificity and sensitivity. However, their cost and complexity remain a significant hurdle to their applications in resource-limited settings. Thus, point-of-need molecular diagnostic platforms that can be used by minimally trained personnel are needed. The nuclease protection assay (NPA) is a nucleic acid hybridization-based technique that does not rely on amplification, can be paired with other methods to improve specificity, and has the potential to be developed into a point-of-need device. In traditional NPAs, hybridization of an anti-sense probe to the target sequence is followed by single-strand nuclease digestion. The double-stranded target-probe hybrids are protected from nuclease digestion, precipitated, and visualized using autoradiography or other methods. We have developed a paper-based nuclease protection assay (PB-NPA) that can be implemented in field settings as the detection approach requires limited equipment and technical expertise. The PB-NPA uses a lateral flow format to capture the labeled target-probe hybrids onto a nitrocellulose membrane modified with an anti-label antibody. A colorimetric enzyme-substrate pair is used for signal visualization, producing a test line. The nuclease digestion of non-target and mismatched DNA provides high specificity while signal amplification with the reporter enzyme-substrate provides high sensitivity. We have also developed an on-chip sample pretreatment step utilizing chitosan-modified paper to eliminate possible interferents from the reaction and preconcentrate nucleic acids, thereby significantly reducing the need for auxiliary equipment. Graphical abstract.

The diagnosis of dengue infection is still a critical factor determining success in the clinical management and treatment of patients. Here, the development of microfluidic paper-based analytical devices (μPADs) utilizing a sandwich immunoassay on wax patterned paper functionalized with anti-dengue NS1 monoclonal antibodies for point-of-care detection of dengue NS1 (DEN-NS1-PAD) is reported. Various assay conditions, including the length of the channel and diluent, were optimized, and the response detected by the naked eye and digitized images within 20–30 min. The DEN-NS1-PAD was successfully tested in the field for detecting dengue NS1 in buffer, cell culture media, and human serum. The limit of detection (LoD) of the DEN-NS1-PAD obtained with the naked eye, scanner, and a smartphone camera was 200, 46.7, and 74.8 ng mL−1, respectively. The repeatability, reproducibility, and stability of the DEN-NS1-PAD were also evaluated. High true specificity and sensitivity in the serum of pediatric patients were observed. These evaluation results confirm that the DEN-NS1-PAD can potentially be used in point-of-care dengue diagnostics, which can significantly impact on the spreading of mosquito-borne diseases, which are likely to become more prevalent with the effects of global warming.

Microfluidic paper-based analytical devices (μPADs) have been widely used in point-of-care testing owing to their simple operation, low volume of the sample required, and the lack of the need for an external force. To obtain accurate semi-quantitative or quantitative results, μPADs need to respond to the challenges posed by differences in reaction conditions. In this paper, multi-layer μPADs are fabricated by the imprinting method for the colorimetric detection of C-reactive protein (CRP). Different lighting conditions and shooting angles of scenes are simulated in image acquisition, and the detection-related performance of μPADs is improved by using a machine learning algorithm. The You Only Look Once (YOLO) model is used to identify the areas of reaction in μPADs. This model can observe an image only once to predict the objects present in it and their locations. The YOLO model trained in this study was able to identify all the reaction areas quickly without incurring any error. These reaction areas were categorized by classification algorithms to determine the risk level of CRP concentration. Multi-layer perceptron, convolutional neural network, and residual network algorithms were used for the classification tasks, where the latter yielded the highest accuracy of 96%. It has a promising application prospect in fast recognition and analysis of μPADs.

Propofol is a widely used intravenous anesthetic agent in sedation and general anesthesia. To improve the safety and maintain the depth of anesthesia, it is important to develop a rapid, sensitive, and reliable method to monitor the concentration of propofol in blood during anesthesia continuously. Here, we present a novel strategy based on paper spray ionization-mass spectrometry (PSI-MS) to detect propofol. Samples (in 10 μL) were mixed with methanol as protein precipitation solvent and 2,6-dimethylphenol as internal standard. Protein micro-precipitation was achieved with methanol by vortexing and centrifuging for 5 s each, and propofol was extracted to the supernatant. PSI-MS was performed in negative ionization mode, and MS signal lasted for 1 min. The analysis of a single sample was completed within 2 min. The area ratios of propofol to internal standard were calculated for quantification. Limit of detection of 5.5 ng mL−1 and limit of quantification of 18.2 ng mL−1 were achieved for propofol in whole blood. Calibration curve was linear in the range of 0.02–10 μg mL−1. The developed method was used successfully in monitoring the propofol concentration in 3 patients’ whole blood during anesthesia, showing its further application in controlling and feeding-back target concentration infusion.

Nowadays, in the clinical, pharmaceutical, and environmental sectors, the development of facile and sensitive analytical methods and/or innovative devices for the follow-up and detection of antibiotics and pharmaceutical formulations, in general, are urgently needed and still challenging. This work declared three vital applications for broad-spectrum nitrofurantoin (macrofuran) antibiotic detection and quantification: A colorimetric method, a coated paper strip-based nano-lanthanum complex prototype and fabrication of smart electronic color sensor device-based coated paper strips. The colorimetric method showed a significant response upon increasing the concentration of the nitrofurantoin in a range between (1.0–100.0 ng/mL) via a visual color change from orange-yellow to red colors degree with detection and quantification limits of 0.175 and 0.53 ng/mL, respectively, whereas the nano-lanthanum complex coated paper strip prototype showed qualitative on-site sensing for nitrofurantoin via naked eye color changes which can be detected anywhere. Moreover, a smart prototype for detecting macrofuran in the means of paper color change in the RGB color component extraction algorithm and the grayscale projection value processing algorithm was fabricated. The change in RGB color on the coated paper strip was detected using an electronic color sensor device. The developed colorimetric method, coated paper strip, and the electronic color sensor device prototype exhibited fast, simple, costless, and selective towards macrofuran over the competing analyzed. As well as, showed good applicability in the different real samples spiked with different concentrations of macrofuran. Graphical abstract

Analytical strategies to ensure the quality and safety of food products without the need for skilled personnel are highly required. The simplicity of glucose strips for diabetes monitoring should be translated to the agri-food sector, ensuring easy evaluation of certain molecules and/or the freshness of a beverage/foodstuff. In addition to the well-known advantages that are characteristic of electroanalytical methods over other methods, paper-based materials are being used to further reduce the gap between complex laboratory testing and simple point-of-need testing. This article highlights some of the most recent advances in the development of paper-based electrochemical approaches for food surveillance, specifically focusing on the use of novel paper-based materials. Two examples are discussed: the development of a miniaturized biosensor realized on copy paper for the quantification of ethanol in commercial beers, and the measurement of ascorbic acid in food supplements within printed electronics supports. Paper-based materials have the potential to lower economic costs, simplify the operative tasks, and most importantly reduce waste generation. The continued combination of manufacturing methods and functional (smart) materials will facilitate the implementation of food analysis at the point of need.

Bacillus cereus is one of the most common foodborne pathogens found in various kinds of staple foods such as rice and wheat. A rapid and accurate detection method for this pathogen is highly desirable for the sustainable production of relevant food products. While several classical and molecular-based detection methods are available for the identification of B. cereus, they suffered one or more limitations such as the requirement for a tedious and time-consuming process, less than ideal specificity, and the lack of portability. Herein, we developed the first paper-based sensing device that exhibits high species specificity with sufficiently low limit of detection for the visual detection of specific DNA sequences of B. cereus. The success is attributed to the strategic planning of fabrication in various dimensions including thorough bioinformatics search for highly specific genes, the use of the pyrrolidinyl peptide nucleic acid (PNA) probe whose selectivity advantage is well documented, and an effective PNA immobilization and DNA-binding visualization method with an internal cross-checking system for validating the results. Testing in rice matrices indicates that the sensor is capable of detecting and distinguishing B. cereus from other bacterial species. Hence, this paper-based sensor has potential to be adopted as a practical means to detect B. cereus in food industries.

We report a smartphone–paper-based sensor impregnated with cetyltrimethylammonium bromide modified silver nanoparticles (AgNPs/CTAB) for determination of Fe3+ in water and blood plasma samples. The methodology for determination of Fe3+ is based on the change in signal intensity of AgNPs/CTAB fabricated on a paper substrate after the deposition of analyte, using a smartphone followed by processing with ImageJ software. The mechanism of sensing for detection and determination of Fe3+ is based on the discoloration of AgNPs which impregnated the paper substrate. The discoloration is attributed to the electron transfer reaction taking place on the surface of NPs in the presence of CTAB. Fe3+ was determined when the paper was impregnated with 1 mM AgNPs for 5 min of reaction time and the substrate was kept under acidic conditions. The linear range for determination of total iron in terms of Fe3+ was 50–900 μg L−1 with a limit of determination (LOD) of 20 μg L−1 and coefficient of variation (CV) of 3.2%. The good relative recovery of 91.3–95.0% and interference studies showed the selectivity of the method for determination of total iron in water and blood plasma samples. Smartphone–paper-based sensors have advantages of simplicity, rapidity, user-friendliness, low cost, and miniaturization of the method for on-site determination of total iron compared to methods that require sophisticated analytical instruments.

Hypochlorous acid (HClO), the core bactericidal substance of the human immune system, plays a vital role in many physiological and pathological processes in the human body. In this work, we designed and synthesized a novel deep-red fluorescent probe TCF-ClO for the determination of hypochlorous acid through theoretical analysis. The results showed that probe TCF-ClO exhibited excellent characteristics of long-wavelength emission (635 nm), fast response ( 1 min), and low detection limit (24 nM). In addition, it had been successfully used for imaging of HClO in living HeLa cells. More importantly, the TCF-ClO composited paper-based sensing material was successfully constructed. The RGB/gray value was obtained from a mobile phone and computer, which could achieve the quantitative detection of HClO, with a linear detection range of 0-50 μM and a detection limit of 1.09 μM (RGB mode)/3.38 μM (gray mode). The function of the paper-based sensor extended from qualitative to quantitative detection of HClO, and it is expected to become a portable device widely used in the environmental area.

The copper(II) ion (Cu2+) has played an indispensable role in diverse kinds of functional physiological processes of organisms, which has become of growing interest. Despite the fact that numerous Cu2+ test papers using fluorescent probes have been fabricated, sensors featuring the ratiometric property that integrates quenched probes and an inner standard dye are rarely reported. Herein, a two-component ratiometric sensor in a paper-based device is proposed to realize highly selective Cu2+ detection. To overcome shortcomings such as low signal-to-noise ratio and incorrect response of the quenching probe, a novel BODIPY-based turn-off probe (P2017) is designed and introduced into the paper-based device with better water solubility and selectivity for Cu2+ detection. Furthermore, a reference dye (B001), exhibiting an emission at 690 nm when the excitation wavelength is 480 nm, is also introduced into the paper-based device. These two components can enhance the quality of the signal as P2017 is sensitively quenched by Cu2+, while B001 with a photostable property, serving as an internal benchmark, is unable to react with Cu2+. The results indicated that the two components provided a new concept for optimizing paper-based device fabrication and developing accurate, simple, and inexpensive Cu2+ detection methods, which could be potentially applied to monitor human health and the environment in remote areas.

Microfluidic paper-based devices (μPADs) and wearable devices have been highly studied to be used as diagnostic tools due to their advantages such as simplicity and ability to provide instrument-free fast results. Diseases such as periodontitis and diabetes mellitus can potentially be detected through these devices by the detection of important biomarkers. This study describes the development of μPADs through craft cutter printing for glucose and nitrite salivary diagnostics. In addition, the use of μPADs integrated into a mouthguard as a wearable sensor for glucose monitoring is also presented. μPADs were designed to contain two detection zones for glucose and nitrite assays and a sampling zone interconnected by microfluidic channels. Initially, the analytical performance of the proposed μPADs was investigated and it provided linear behavior (r2 ≥ 0.994) in the concentration ranges between 0 to 2.0 mmol L−1 and 0 to 400 μmol L−1 for glucose and nitrite, respectively. Under the optimized conditions, the limits of detection achieved for glucose and nitrite were 27 μmol L−1 and 7 μmol L−1, respectively. Human saliva samples were collected from healthy individuals and patients previously diagnosed with periodontitis or diabetes and then analyzed on the proposed μPADs. The results found using μPADs revealed higher glucose concentration values in saliva collected from patients diagnosed with diabetes mellitus and greater nitrite concentrations in saliva collected from patients diagnosed with periodontitis, as expected. The results obtained on μPADs did not differ statistically from those measured by spectrophotometry. With the aim of developing paper-based wearable sensors, μPADs were integrated, for the first time, into a silicone mouthguard using a 3D-printed holder. The proof of concept was successfully demonstrated through the monitoring of the glucose concentration in saliva after the ingestion of chocolate. According to the results reported herein, paper-based microfluidic devices offer great potential for salivary diagnostics, making their integration into a silicone mouthguard possible, generating simple, low-cost, instrument-free, and powerful wearable sensors.

This manuscript reports on a simple paper-based bienzymatic colorimetric assay for sarcosine as an important urinary biomarker of prostate cancer. All required assay reagents are pre-deposited on hydrophilic filter paper spots surrounded by a hydrophobic barrier. Sarcosine in the sample solution is selectively oxidized in the presence of sarcosine oxidase (SOx), resulting in the formation of hydrogen peroxide, which is subsequently detected through the horseradish peroxidase (HRP)–catalyzed conversion of the colorless indicator 3,3’,5,5’-tetramethylbenzidine (TMB) into its blue-colored oxidation product. By the modification of the paper with positively charged poly(allylamine hydrochloride) (PAH), a linear response to sarcosine between 0 and 10 μM and a significant lowering of the limit of detection (LOD) (0.6 μM) compared to the unmodified paper substrate (12.6 μM) has been achieved. The improvement of the LOD was attributed to the fact that the presence of the polymer limits the enzyme-driven colorimetric reaction to the surface of the paper substrate, resulting in stronger color development. In experiments in artificial urine matrix, the bicarbonate anion was identified as an inhibitor of the colorimetric reaction. This inhibition was successfully eliminated through on-device sample pH adjustments with pH-buffer components pre-deposited onto assay devices. The LOD for sarcosine achieved in artificial urine matrix (2.5 μM) is below the 5 μM threshold value for this urinary biomarker required for diagnostic purposes. Finally, good selectivity over all 20 natural amino acids and satisfactory long-term storage stability of reagent-modified paper substrates at − 20 °C for a period of 50 days were confirmed.

Infections caused by foodborne microorganisms are a great threat to the global environment and public healthcare today. Thus, rapid, portable and sensitive assays that can realize the identification of foodborne bacteria are highly desired. In this study, a smart fluorescent and colorimetric dual-readout sensing system has been established for simple and rapid E. coli determination by utilizing the Cu2+-triggered oxidation of o-phenylenediamine (OPD). Initially, Cu2+ can oxidize OPD to OPDox, resulting in an orange-yellow fluorescence and visible pale-yellow color. However, E. coli can effectively reduce Cu2+ into Cu+, inhibiting the Cu2+-triggered oxidation of OPD to OPDox. Consequently, the introduction of E. coli can turn off both the fluorescence and the UV-vis absorbance signals of the OPD-Cu2+ system, illustrating an original mechanism for fluorescent and colorimetric dual-channel detection of E. coli. Moreover, a filter paper-based visual sensor was built and coupled with OPD-Cu2+ solution under the assistance of a UV lamp. The as-prepared sensor can detect E. coli quantitatively with the help of a typical smartphone color-scanning application (APP). Thus, this study offers a valid dual-mode assay for sensitive and on-site visible detection of E. coli, guaranteeing the reliability of the results and is more attractive for practical use. Graphical Abstract Schematic illustration of the smartphone-integrated sensing system for fluorescent and colorimetric dual-channel detection of E. coli based on the Cu2+-OPD system.

A method named imprint paper spray ionization mass spectrometry (imprint-PSI-MS) has been developed and employed for the determination of pungency of peppers. A pepper fruit was cut into a triangular shape, deposited onto a triangular paper, and compressed by a homemade press tool aiming to imprint and transfer the pepper constituents onto the paper surface. Subsequently, the triangular paper was submitted to conventional PSI-MS analysis. Twelve peppers were analyzed, ranging from highly pungent to lowly pungent taste. Pepper pungency values from the Scoville scale (in Scoville heat units, SHU) were compared with the ion intensities of the capsaicin and dihydrocapsaicin compounds obtained from the imprint-PSI-MS analysis, and a correlation coefficient of 0.97 was achieved. In addition, the ion intensities of a sugar compound were monitored in all peppers, and the results were compared with the Scoville scale. Low sugar ion intensities were detected in pungent peppers, while high ion intensities were achieved in low-pungent peppers, suggesting that the pepper pungency may be determined by inversely relating pungency to sugar contents. This work demonstrates the utility of the imprint-PSI-MS method to perform rapid qualitative analyses of peppers and estimate the pungency by monitoring the pepper metabolites. Graphical abstract.

Simple and visual quantitative detection of foodborne pathogens can effectively reduce the outbreaks of foodborne diseases. Herein, we developed a simple and sensitive quantum dot (QD)-based paper device for visual and quantitative detection of Escherichia coli (E. coli) O157:H7 based on immunomagnetic separation and nanoparticle dissolution-triggered signal amplification. In this study, E. coli O157:H7 was magnetically separated and labeled with silver nanoparticles (AgNPs), and the AgNP labels can be converted into millions of Ag ions, which subsequently quench the fluorescence of QDs in the paper strip, which along with the readout can be visualized and quantified by the change in length of fluorescent quenched band. Owing to the high capture efficiency and effective signal amplification, as low as 500 cfu mL−1 of E. coli O157:H7 could be easily detected by naked eyes. Furthermore, this novel platform was successfully applied to detect E. coli O157:H7 in spiked milk samples with good accuracy, indicating its potential in the detection of foodborne pathogens in real samples.

We develop a capillary-paper spray (CPS) ion source which allows for sample separation in the capillary and enables rapid and sensitive paper spray (PS) mass spectrometry (MS) analysis of biofluids. The CPS employs a glass capillary to load liquid analytes, vertically standing at the rear of the PS. To further reduce the matrix effect, a nitrocellulose filter membrane is placed between the glass tube and chromatography paper to absorb proteins and other macromolecules, which is beneficial for the detection of the small molecules. Compared with the normal PS method, the CPS method markedly improves spray stability and prolongs analysis duration, and also generates significantly better signal intensities during the analysis of drugs, thus indicating its potential for clinical use. As a proof of concept, quantitative analysis of drugs (metformin hydrochloride and berberine hydrochloride) in serum is performed.

Ultrasensitive detection of specific biomarkers in clinical serum is helpful for early diagnosis of cervical cancer. In this paper, a surface-enhanced Raman scattering (SERS)-based immunoassay was developed for the simultaneous determination of squamous cell carcinoma antigen (SCCA) and osteopontin (OPN) in cervical cancer serum. Au-Ag nanoshuttles (Au-AgNSs) as SERS tags and hydrophobic filter paper-based Au nanoflowers (AuNFs) as capture substrate were constructed into a sandwich structure which served as an ultrasensitive SERS-based immunoassay platform. Finite difference time domain simulation confirmed that the electromagnetic field coupled between the AuNFs had a prominent SERS signal enhancement effect, which improved the detection sensitivity. SERS mapping showed that hexadecenyl succinic anhydride hydrophobic treatment could prevent the analyte from being quickly absorbed by the filter paper and increase the retention time to be more evenly distributed on the filter paper substrate. The immunoassay platform was verified to have good selectivity and reproducibility. With this method, the detection limits of SCCA and OPN in human serum were as low as 8.628 pg/mL and 4.388 pg/mL, respectively. Finally, in order to verify the feasibility of its clinical application, the serum samples of healthy subjects; cervical intraepithelial neoplasia I (CINI), CINII, and CINIII; and cervical cancer patients were analyzed, and the reliability of the results was confirmed by enzyme-linked immunosorbent assay experiments. The constructed SERS-based immunoassay platform could be used as a clinical tool for early screening of cancers in the future.

Respiratory viral infections often mimic the symptoms of infections caused by bacteria; however, restricted and targeted administration of antibiotics is needed to combat growing antimicrobial resistance. This is particularly relevant in low-income settings. In this work, we describe the use of isothermal amplification of viral DNA at 37 °C coupled to a paper-based vertical flow microarray (VFM) setup that utilizes a colorimetric detection of amplicons using functionalized gold nanoparticles. Two oligonucleotide probes, one in-house designed and one known adenoviral probe were tested and validated for microarray detection down to 50 nM using a synthetic target template. Furthermore, primers were shown to function in a recombinase polymerase amplification reaction using both synthetic template and viral DNA. As a proof-of-concept, we demonstrate adenoviral detection with four different adenoviral species associated with respiratory infections using the paper-based VFM format. The presented assay was validated with selected adenoviral species using the in-house probe, enabling detection at 1 ng of starting material with intra- and inter-assay %CV of ≤ 9% and ≤ 13%. Finally, we validate our overall method using clinical samples. Based on the results, the combination of recombinase polymerase amplification, paper microarray analysis, and nanoparticle-based colorimetric detection could thus be a useful strategy towards rapid and affordable multiplexed viral diagnostics. Electronic supplementary material The online version of this article (10.1007/s00216-018-1503-y) contains supplementary material, which is available to authorized users.

Rapid and reliable identification of bacteria is an important issue in food, medical, forensic, and environmental sciences; however, conventional procedures are time-consuming and often require extensive financial and human resources. Herein, we present a label-free method for bacterial discrimination using surface-enhanced Raman spectroscopy (SERS) and partial least squares discriminant analysis (PLS-DA). Filter paper decorated with gold nanoparticles was fabricated by the dip-coating method and it was utilized as a flexible and highly efficient SERS substrate. Suspensions of bacterial samples from three genera and six species were directly deposited on the filter paper-based SERS substrates before measurements. PLS-DA was successfully employed as a multivariate supervised model to classify and identify bacteria with efficiency, sensitivity, and specificity rates of 100% for all test samples. Variable importance in projection was associated with the presence/absence of some purine metabolites, whereas confidence intervals for each sample in the PLS-DA model were calculated using a resampling bootstrap procedure. Additionally, a potential new species of bacteria was analyzed by the proposed method and the result was in agreement with that obtained via 16S rRNA gene sequence analysis, thereby indicating that the SERS/PLS-DA approach has the potential to be a valuable tool for the discovery of novel bacteria. Graphical abstract This paper describes the discrimination of bacteria at the genus and species levels, after minimal sample preparation, using paper-based SERS substrates and PLS-DA with uncertainty estimation.

Speciation of chromium (Cr) was demonstrated using microfluidic paper-based analytical devices (μ-PADs) that permit the colorimetric determination of hexavalent chromium (Cr(VI)) and trivalent chromium (Cr(III)) via online oxidation. The μ-PADs consist of left and right channels that allow the simultaneous measurements of Cr(VI) and total Cr based on the colorimetric reaction of Cr(VI) with 1,5-diphenylcarbazide (DPC). For the determination of Cr(VI), a sample solution was directly reacted with DPC in the left channels whereas total Cr was determined in the right channels, which permitted online oxidation in the pretreatment zone containing cerium (IV) (Ce(IV)) followed by a colorimetric reaction with DPC. We found that the online oxidation of Cr(III) proceeded 100% whereas Ce(IV) inhibited the reaction of Cr(VI) with DPC. Therefore, speciation can be achieved by measuring the Cr(VI) and total Cr in the left and right channels followed by the subtraction of Cr(VI) from total Cr. The limits of detection and quantification were 0.008 and 0.02 mg L−1 for Cr(VI) and 0.07 and 0.1 mg L−1 for Cr(III) or total Cr, respectively. The linear dynamic ranges were 0.02–100 mg L−1 and 0.1–60 mg L−1 for Cr(VI) and Cr(III), respectively. The RSDs were less than 7.5%. The results obtained using μ-PADs were in good agreement with those obtained via ICP-OES with recoveries of 92–108% for Cr(III) and 108–110% for Cr (VI) using μ-PADs, and 106–110% for total Cr using ICP-OES. Thus, the μ-PADs could potentially be utilized for the speciation of chromium in developing countries where environmental pollution and the availability of sophisticated instruments are significant problems.

Microfluidic-based biosensors have been developed for their precise automatic reaction control. However, these biosensors require external devices that are difficult to transport and use. To overcome this disadvantage, our group made an easy-to-use, cheap, and light pumpless three-dimensional photo paper–based microfluidic analytical device (3D-μPAD; weight: 1.5 g). Unlike conventional paper-based microfluidic analytical devices, the 3D-μPAD can be used to control fluid flow in a 3D manner, thus allowing sophisticated multi-step reaction control. This device can control fluid flow speed and direction accurately using only the capillary-driven flow without an external device like a pump. The flow speed is controlled by the width of the microfluidic channel and its surface property. In addition, fluid speed control and 3D-bridge structure enable the control of fluid flow direction. Using these methods, multi-step enzyme-linked immunosorbent assay (ELISA) can be done automatically in sequence by injecting solutions (sample, washing, and enzyme’s substrate) at the same time in the 3D-μPAD. All the steps can be performed in 14 min, and data can be analyzed immediately. To test this device, thioredoxin-1 (Trx-1), a biomarker of breast cancer, is used as the target. In the 3D-μPAD, it can detect 0–200 ng/mL of Trx-1, and the prepared 3D-μPAD Trx-1 sensor displays excellent selectivity. Moreover, by analyzing the concentration of Trx-1 in real patients and healthy individuals’ blood serum samples using the 3D-μPAD, and comparing results to ELISA, it can be confirmed that the 3D-μPAD is a good tool for cancer diagnosis.

On-site rapid monitoring of nitrite as an assessment indicator of the environment, food, and physiological systems has drawn extensive attention. Here, electrokinetic stacking (ES) was combined with colorimetric reaction on a paper-based device (PAD) to achieve colorless nitrite detection with smartphone. In this paper, nitrite was stacked on the paper fluidic channel as a narrow band by electrokinetic stacking. Then, Griess reagent was introduced to visualize the stacking band. Under optimal conditions, the sensitivity of nitrite was 160-fold increased within 5 min. A linear response in the range of 0.075 to 1.0 μg mL−1 (R2 = 0.99) and a limit of detection (LOD) of 73 ng mL−1 (0.86 μM) were obtained. The LOD was 10 times lower than the reported PAD, and close to that achieved by a desktop spectrophotometer. The applicability was demonstrated by nitrite detection from saliva and water with good selectivity, adding 100 times more concentrated co-ions. High recovery (91.0~108.7%) and reasonable intra-day and inter-day reproducibility (RSD

Paper-based immunoassays are effective methods that employ microfluidic paper-based analytical devices (μPADs) for the rapid, simple, and accurate quantification of analytes in point-of-care diagnosis. In this study, we developed a wax-printed multilayered μPAD for the colorimetric detection of carcinoembryonic antigen (CEA), where the device contained a movable and rotatable detection layer to allow the μPAD to switch the state of the sample solutions, i.e., flowing or storing in the sensing zones. A smartphone with a custom-developed program served as an automated colorimetric reader to capture and analyze images from the μPAD, before calculating and displaying the test results. After optimizing the crucial conditions for the assay, the proposed method exhibited a wide linear dynamic range from 0.5 to 70 ng/mL, with a low CEA detection limit of 0.015 ng/mL. The clinical performance of this method was successfully validated using 50 positive and 40 negative human serum samples, thereby demonstrating the high sensitivity of 98.0% and specificity of 97.5% in the detection of CEA. The proposed method is greatly simplified compared with the cumbersome steps required for traditional immunoassays, but without any loss of accuracy and stability, as well as reducing the time needed to detect CEA. Complex and bulky instruments are replaced with a smartphone. The proposed detection platform could potentially be applied in point-of-care testing. Graphical abstract.

Carbamazepine is an antiepileptic drug with a narrow therapeutic index, which requires an efficient method for blood level monitoring. Finger-prick dried blood spot (DBS) collection is an alternative microsampling technique, which is less invasive than conventional venipuncture. Paper-based molecularly imprinted-interpenetrating polymer networks (MI-IPN) were developed as blood collection devices, which allowed for selective on-spot microextraction of carbamazepine from DBS. A hybrid of homogeneous polystyrene and silica gel polymer was synthesized and coated on a Whatman® Grade 1 filter paper. Proteins and other interferences in the blood samples were eliminated by using the MI-IPN collection devices, and the resulting DBS extracts were suitable for direct injection into the capillary electrophoretic instrument. The lower limit of quantitation of 4 μg/mL in capillary blood was achieved by the sweeping-micellar electrokinetic chromatography method using a KCl-containing matrix, which was sufficient for the therapeutic drug monitoring purposes. Method accuracies were in the range of 88.4 ± 4.5% to 94.5 ± 2.7% with RSD values ≤ 5.1%. The developed paper-based MI-IPN provided superior extraction efficiencies (92.2 ± 2.5%) in comparison with commercially available DBS collection cards, i.e., Whatman® 903 protein saver card (59.8 ± 2.8%) and GenCollect™ 2.0 card (47.2 ± 1.4%). The paper-based MI-IPN devices for DBS collection and on-spot extraction were characterized by simple fabrication, low costs, disposability, and reduction in sample preparation steps, and their further developments might open new perspectives in clinical applications, such as in therapeutic drug monitoring.

Isoelectric focusing (IEF), a powerful technique for protein separation and enrichment, was successfully integrated into microfluidic paper-based analytical devices (μPADs) in this work. The μPADs for isoelectric focusing were fabricated by octadecyltrichlorosilane (OTS) silanization and subsequent region-selective plasma treatment. The system of IEF on μPADs could be easily assembled. And a series of conditions of the system were investigated, including the suitable concentration of ampholyte to create good pH gradient, the effect of polyvinylpyrrolidone (PVP) on electroosmotic flow (EOF) suppression, and focusing voltage applied on the paper channel. After optimization, simultaneous separation and enrichment of protein sample containing myoglobin and cytochrome C was successfully demonstrated. Besides, parallel IEF on multichannels were also achieved for the separation of multiple protein samples on one single chip, and their performance was compared with that of the conventional gel-IEF system. The developed IEF on μPADs exhibits appealing features such as low cost, simplicity, and disposability and are believed to have great application potentials.

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.

Chemiluminescence (CL) reagent luminol was loaded into the porous structure of cobalt-imidazole metal-organic framework (MOF) ZIF-67 to obtain luminol-functionalized ZIF-67 (luminol@ZIF-67) with CL property. The morphology, composition, CL property, and CL mechanism of luminol@ZIF-67 were carefully investigated. The obtained luminol@ZIF-67 exhibited strong, stable, and visible CL emission that reacted with H2O2, attributed to the strong catalytic effect of ZIF-67 combined with the shortened diffusion distance between luminol and the catalytic center. The CL intensity of luminol@ZIF-67 was more than 550 times higher than that of luminol. Catechol can effectively quench the CL emission of luminol@ZIF-67 that reacted with H2O2. Then, a simple paper-based CL imaging detection method was developed for the detection of catechol by using a smartphone as a portable detector. The linear calibration curve of the developed CL assay for catechol ranged from 5 to 100 mg/L with detection limit of 1.1 mg/L (S/N = 3δ). The strong CL emission of luminol@ZIF-67 combined with the effective quench ability of catechol guaranteed high sensitivity of the detection method. The practical application ability of the developed CL assay was tested by the determination of catechol in tea and tap water samples, resulting in acceptable results. This work provides an effective paper-based CL detection method for catechol and enriches the species of the chemiluminescent MOF material.

A colorimetric paper-based enzyme-coupled antimony tin oxide nanoparticle (ATONP) nanobiosensor for selective detection of Cd2+ ions in clams and mussels is presented. Alkaline phosphatase (ALP) was immobilized on ATONPs via 16-phosphonohexadecanoic acid (16-PHA) to develop ATONP-ALP nanobiosensor. The biosensor was characterized using XPS, Raman spectroscopy, SEM, and EDX. ATONP-ALP nanobiosensor exhibited high selectivity towards detection of Cd2+ ion with a LOD 0.006 μg L−1 and linear range of detection 0.005–1 μg L−1. The developed biosensor was further integrated into a low-cost paper-based format. A visual color change was obtained for Cd2+ ion in the range 0.1–10 μg L−1. The developed biosensor was successfully demonstrated for the analysis of Cd2+ ions in clams with recoveries 101–104%. The ATONP-ALP nanobiosensor was validated using mussel tissue (BCR-668) and the conventional ICP-OES and ICP-MS techniques.

We describe the use of a chemometrics-based computational platform to optimize a glucose assay on a microfluidic paper-based analytical device (μPAD). Glucose is colorimetrically detected in the presence of glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI). Using a Y-shaped paper microfluidic chip, the concentration of glucose, volume of reagents, and the length and width of the microfluidic channel were examined. The responses of the microfluidic chips were analyzed at the halfway point of the channel length. Variables affecting the response were screened by using a 24 factorial design, and among them, volume and concentration of the glucose were optimized by applying a rotatable central composite design (CCD). The optimum and experimental responses are 151.58 and 149.80, respectively, with an absolute error of 1.2%.

A rapid in situ analytical method was developed for the detection of generated carcinogenic aromatic amines from banned azo dyes utilizing a photocatalytic reduction-based liquid microjunction surface sampling (LMJSS)–mass spectrometry (MS) system. We utilized photocatalytic reduction under UV irradiation with TiO2 as catalyst to have rapid and mild reduction of azo dyes. The reaction conditions were optimized to have complete photocatalytic reduction within 2–5 min in pure methanol at room temperature. TiO2 was immobilized in the inner wall of the capillaries in the LMJSS system to achieve in situ sampling–online rapid reduction–MS detection for aromatic amines originating from azo dyes in packaging surface. The yields of in-tube photocatalytic reduction were near 100% by delivering the azo dye extracts through the capillary at 1 μL/min under UV irradiation. With this design, in situ analysis was completed within 2 min via direct MS detection and 7 min via liquid chromatography (LC)–MS detection. The detection limits for five aromatic amines originating from four different azo dyes were in the range of 1–17 mg/kg with relative standard deviations (RSDs)

A rapid and low-cost method of diagnosis is becoming important for detecting fetal inherited diseases, including single-gene disorders and chromosomal abnormalities. Here, we demonstrated an innovation that use paper-dried cord blood (PCB) as the starting material for PCR and whole genome amplification without any DNA extraction step at a very low cost. A novel PCR buffer named "DDB buffer" containing ammonium sulfate and glycerol were used instead of the conventional 10× PCR buffer. The amplicons were directly analyzed through microchip electrophoresis and whole genome sequencing. Inhibitory substances in filter paper were effectively inactivated using DDB buffer. Direct PCR amplification of DNA fragments ranging from 100 to 900 bp using filter paper spotted with 0.5 to 5 μL of cord blood and various anticoagulants was successful. We were able to determine fetal single-gene disorders and chromosomal diseases in all 46 chromosomes using PCB samples successfully. Compared with prenatal diagnosis using purified DNA, the proposed method is simple, fast, less prone to cross-contamination at minimal cost. Researchers and clinical and healthcare workers may employ this method for genetic diagnosis using cord blood samples with minimum laboratory resources. This method is very promising for a variety of genetic diagnosis applications in underserved communities at the point of need in developing areas. Graphical abstract.

Lateral flow type detection is becoming interesting not only in regions with a poor medical infrastructure but also for practitioners in day-to-day clinical work or for veterinary control in case of possible epidemics. In this work, we describe the first steps of development of a multi-channel strip with potential internal calibration of multiparametric and colorimetric lateral flow assays for the simultaneous detection of the lipopolysaccharides (LPS) of Salmonella typhimurium (S. typhimurium) and Salmonella enteritidis (S. enteritidis). We structured four channels in the nitrocellulose membrane with a Yb:KGW solid-state femtosecond laser ("cold" ablation process) to form distinct tracks of porous material and used gold nanoparticles for the labeling of the antibodies. In addition, calibration curves of the spot intensities of both serovars are presented, and it was shown that no cross reactivity between the different capture antibodies and LPS occurred. Finally, we detected LPS of both Salmonella serovars simultaneously. The color changes (spot intensities of the reaction zones) were evaluated using the open-source image-processing program ImageJ. Graphical abstract Multiparametric testing, strip A was tested with LPS S. enteritidis ( c=0.01 g/L) and LPS S.typhimurium ( c=0.0001 g/L), strip B with LPS S. enteritidis ( c=0.001 g/L) and LPS S. typhimurium ( c=0.001g/L) and strip C with LPS S. enteritidis (c=0.0001 g/L) and LPS S. typhimurium ( c=0.01 g/L), and read-out.

Mitigation of the peroxide explosive threat, specifically triacetone triperoxide (TATP) and hexamethylene triperoxide diamine (HMTD), is a priority among the law enforcement community, as scientists and canine (K9) units are constantly working to improve detection. We propose the use of paper spray ionization–high-resolution mass spectrometry (PSI-HRMS) for detection of peroxide explosives in biological matrices. Occurrence of peroxide explosives and/or their metabolites in biological samples, obtained from urine or blood tests, give scientific evidence of peroxide explosives exposure. PSI-HRMS promote analysis of samples in situ by eliminating laborious sample preparation steps. However, it increases matrix background issues, which were overcome by the formation of multiple alkali metal adducts with the peroxide explosives. Multiple ion formation increases confidence when identifying these peroxide explosives in direct sample analysis. Our previous work examined aspects of TATP metabolism. Herein, we investigate the excretion of a TATP glucuronide conjugate in the urine of bomb-sniffing dogs and demonstrate its detection using PSI from the in vivo sample.

Herein we report a quantitative, multiplex assay for disease markers in plasma based on an integrated setup of a portable scanner and a disposable paper-based analytical device (PAD). The quantitative analysis relies on the digital colorimetric reading of the three-layer PAD with 30 assay sites for performing respective chromogenic reactions for plasma uric acid, glucose, and triglyceride, which are considered as important risk factors for cardiovascular diseases. A portable scanner with WiFi transmission capability was used to produce high-quality color images of the PADs and wirelessly transfer them to a smartphone or other mobile devices for data processing. The concentrations of biomarkers in both standard solutions and plasma samples can be directly obtained using a custom-designed smartphone app that is also capable of constructing calibration curves. The detection limits of uric acid, glucose, and triglyceride were determined to be 0.50 mg/dL, 0.84 mmol/L, and 14 mg/dL, respectively, which are below the normal limits and adequate for clinical validation. Owing to the distinct advantages—simple, portable, and cost-effective—this mobile assay protocol can be used for point-of-care (POC) settings or resource-limited situations, and potentially for the diagnosis and prevention of infectious diseases.

Paper-based analytical devices (PADs) have encountered a wealth of applications in recent years thanks to the numerous advantages of paper as a support. A silver nanoflower (AgNF) modified paper-based dual substrate for both surface-enhanced Raman spectroscopy (SERS) and ambient pressure paper spray mass spectrometry (PS-MS) was developed. AgNFs were immobilized on nylon-coated paper modified with silver and ethylenediamine. The developed substrate was characterized via scanning electron microscopy and infrared spectroscopy. The densely packed nanoscale petals of the AgNFs lead to a large number of so-called hot spots at their overlapping points, which result in an enhancement of the Raman signal. In addition, the presence of the AgNFs produces an increase in the sensitivity of the mass spectrometric analysis as compared with bare paper and nylon/Ag-coated paper. The dual substrate was evaluated for the identification and quantification of ketoprofen in aqueous standards as well as human saliva from healthy volunteers. The method enables the determination of ketoprofen with a limit of detection and limit of quantification via PS-MS of 0.023 and 0.076 mg L−1, respectively, with a relative standard deviation (RSD) of 3.4% at a concentration of 0.1 mg L−1. This dual substrate enables the simple and fast detection of ketoprofen with minimal sample preparation, providing complementary Raman and mass spectrometric information.