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Microfluidic paper-based analytical devices (μPADs) are emerging as powerful analytical platforms in clinical diagnostics, food safety, and environmental protection because of their low cost and favorable substrate properties for biosensing. However, the existing top-down fabrication methods of paper-based chips suffer from low resolution (200 μm). Additionally, papers have limitations in their physical properties (e.g., thickness, transmittance, and mechanical flexibility). Here, we demonstrate a bottom-up approach for the rapid fabrication of heterogeneously controlled paper-based chip arrays. We simply print a wax-patterned microchip with wettability contrasts, enabling automatic and selective assembly of cellulose microfibers to construct predefined paper-based microchip arrays with controllable thickness. This paper-based microchip printing technology is feasible for various substrate materials ranging from inorganic glass to organic polymers, providing a versatile platform for the full range of applications including transparent devices and flexible health monitoring. Our bottom-up printing technology using cellulose microfibers as the starting material provides a lateral resolution down to 42 ± 3 μm and achieves the narrowest channel barrier down to 33 ± 2 μm. As a proof-of-concept demonstration, a flexible paper-based glucose monitor is built for human health care, requiring only 0.3 μL of sample for testing.

Microfluidic paper-based analytical devices (μPADs) are emerging as prominent platform for disease detection, specifically in developing countries. This paper device offers simplicity and affordability not typically seen in centralized laboratory settings. However, detection limits in μPADs are inadequate and often require tests results to be read within a specific time interval to ensure accuracy. To overcome these challenges, we are developing an on-chip mass spectrometry (MS) detection strategy for immunoassays performed on paper substrates. Herein, we present our initial results from a proof-of-concept study toward the development of μPADs capable of storing immunoassay reagents within the confinements of the 3D device, automatic splitting of biofluid into four individual test zones, immuno-capture of disease biomarker, and on-chip MS detection of captured species. The reported study encourages the development of point-of-care and direct-to-customer testing using the disposable μPAD to collect samples, followed by sensitive analysis using portable mass spectrometers. We demonstrate this capability using malaria Plasmodium falciparum histidine-rich protein 2 (PfHRP2) antigen detection.

Target-responsive nanomaterials attract growing interest in the application of drug delivery, bioimaging, and sensing due to the responsive releasing of guest molecules by the smart molecule gate. However, it remains a challenge to develop smart nanomaterials with simple assembly and low nonspecific leakage starting from encapsulation strategies, especially in the sensing field. Herein, Au nanoclusters (Au NCs) were first grown on porous carbon derived from ZIF-8 (PCZIF) to be employed as nanocarriers. By employing the Au NCs as linkers and aptamer (Apta) double-strand hybrids (target Apta and SH-complementary DNA) as capping units, we reported the novel target-responsive nanomaterials of Apta/Au NCs-PCZIF/hemin through Au-S binding encapsulation for sensing assays. The Au-S binding encapsulation strategy simplified the packaging procedure and reduced non-target responsive leakage. As a proof, ochratoxin A (OTA) as a model target participates in the double-strand hybrid competitive displacement reaction and triggered Apta conformation switches from a coil to a G-quadruplex structure accompanied by the dissociation of the gatekeeper. Simultaneously, the released hemin can initiate a self-assembly to form G-quadruplex/hemin DNAzyme. Interestingly, owing to DNAzyme providing electron transfer mediators and peroxidase-like activity, we proposed an electrochemical/colorimetric dual-mode paper-based analytical device (PAD) that provided self-verification to enhance reliability and accuracy, benefiting from independent signal conversion and transmission mechanism. As a consequence, the proposed dual-mode PAD could achieve sensitive electrochemical detection and visual prediction of OTA in the range of 1 pg/mL to 500 ng/mL and 50 pg/mL to 500 ng/mL, respectively. The electrochemical detection limit for OTA was as low as 0.347 pg/mL (

Illicit drug use causes over half a million deaths worldwide every year. Drugs of abuse are commonly smuggled through customs and border checkpoints and, increasingly, through parcel delivery services. Improved methods for detection of trace drug residues from surfaces are needed. Such methods should be robust, fieldable, sensitive, and capable of detecting a wide range of drugs. In this work, commercially produced paper with a pressure-sensitive adhesive coating was utilized for the collection and analysis of trace drug residues by paper spray mass spectrometry (MS). This modified substrate was used to combine sample collection of drug residues from surfaces with rapid detection using a single paper spray ticket. The all-in-one ticket was used to probe different surfaces commonly encountered in forensic work including clothing, cardboard, glass, concrete, asphalt, and aluminum. A total of 10 drugs (acetyl fentanyl, fentanyl, clonazolam, cocaine, heroin, ketamine, methamphetamine, methylone, U-47700, and XLR-11) were evaluated and found to be detectable in the picogram range using a benchtop mass spectrometer and in the low nanogram range using a portable ion trap MS. The novel approach demonstrates a simple yet effective sampling strategy, allowing for rapid identification from difficult surfaces via paper spray mass spectrometry.

This paper introduces a novel potential-resolved paper-based biosensor for simultaneous detection of multiple microRNAs (miRNAs) (taking miRNA-155 and miRNA-126 as examples) based on the bipolar electrode (BPE) electrochemiluminescence (ECL) strategy. The proposed multiple-channel paper-based sensing microfluidic platform was prepared by wax-printing technology, screen-printing method, and in situ Au nanoparticles (AuNPs) growth to form hydrophilic areas, hydrophobic boundaries, waterproof electronic bridge, driving electrode regions, and parallel bipolar electrode regions. CdTe quantum dots (QDs)-H2 and Au@g-C3N4 nanosheets (NSs)-DNA1 were used as dual electrochemiluminescence signal probes, and carboxylated Fe3O4 magnetic nanoparticles existed as carriers. CdTe QDs-H2/S2O82- and Au@g-C3N4 NSs-DNA1/S2O82- could exhibit two strong and stable ECL emissions at a drive voltage of 9 and 12 V, respectively, which can be used as effective potential-resolved signal tags. In addition, the proposed three-dimensional (3D) DNA nanomachine model and the target miRNA cycle strategy were used to achieve double amplification of electrochemiluminescence intensity. More importantly, the combination of the bipolar electrode system and the potential-resolved multitarget electrochemiluminescence method can greatly reduce the spatial interference between substances. The prepared ECL biosensor showed a favorable linear response for the detection of miRNA-155 and miRNA-126 with relatively low detection limits of 5.7 and 4.2 fM, respectively. With excellent sensitivity, the strategy may provide an efficient method for clinical application, especially in detection of trace multiple targets.

Tuberculosis (TB), one of the deadliest infectious diseases, is caused by Mycobacterium tuberculosis (MTB) and remains a public health problem nowadays. Conventional MTB DNA detection methods require sophisticated infrastructure and well-trained personnel, which leads to increasing complexity and high cost for diagnostics and limits their wide accessibility in low-resource settings. To address these issues, we have developed a low-cost photothermal biosensing method for the quantitative genetic detection of pathogens such as MTB DNA on a paper hybrid device using a thermometer. First, DNA capture probes were simply immobilized on paper through a one-step surface modification process. After DNA sandwich hybridization, oligonucleotide-functionalized gold nanoparticles (AuNPs) were introduced on paper and then catalyzed the oxidation reaction of 3,3',5,5'-tetramethylbenzidine (TMB). The produced oxidized TMB, acting as a strong photothermal agent, was used for the photothermal biosensing of MTB DNA under 808 nm laser irradiation. Under optimal conditions, the on-chip quantitative detection of the target DNA was readily achieved using an inexpensive thermometer as a signal recorder. This method does not require any expensive analytical instrumentation but can achieve higher sensitivity and there are no color interference issues, compared to conventional colorimetric methods. The method was further validated by detecting genomic DNA with high specificity. To the best of our knowledge, this is the first photothermal biosensing strategy for quantitative nucleic acid analysis on microfluidics using a thermometer, which brings fresh inspirations on the development of simple, low-cost, and miniaturized photothermal diagnostic platforms for quantitative detection of a variety of diseases at the point of care.

Multilayered and stacked cellulose paper has emerged as a promising platform for construction of three-dimensional (3D) cell culture because of its low cost, good biocompatibility, and high porosity. However, its poor light transmission makes it challenging to directly and clearly monitor cell behaviors (e.g., growth and proliferation) on the paper-based platform using an optical microscope. In this work, we developed a transparent microcrystalline cellulose/polyvinyl alcohol (MCC/PVA) paper with irregular pores through dissolution and regeneration of microcrystalline nanocellulose, addition of a porogen reagent (NaCl), and subsequently dipping in PVA solutions. The transparent MCC paper displays high porosity (up to 90%), adjustable pore size (between 23 and 46 μm), large thickness (from 315 to 436 μm), and high light transmission under water (95%). Through further modification of the transparent MCC paper with PVA, the obtained transparent MCC/PVA paper shows enhanced mechanical properties (dry and wet strengths), good hydrophilicity (with a contact angle of 70.8°), and improved biocompatibility (cell viability up to 90%). By stacking and destacking multiple layers of the transparent MCC/PVA paper, it has been used for both two-dimensional and three-dimensional cell culture platforms. The transparent MCC/PVA paper under water enables both direct observation of cell morphology by an optical microscope via naked eyes and fluorescence microscope after staining. We envision that the developed transparent MCC/PVA paper holds great potential for future applications in various bioanalytical and biomedical fields, such as drug screening, tissue engineering, and organ-on-chips.

Diabetes mellitus is one of the most common chronic diseases worldwide. Generally, the levels of fasting or postprandial blood glucose and other biomarkers, such as glycated albumin, glycated hemoglobin, and 1,5-anhydroglucitol, are used to diagnose or monitor diabetes progression. In the present study, we developed a sensor to simultaneously detect the glucose levels and glycation ratios of human serum albumin using a lateral flow assay. Based on the specific enzymatic reactions and immunoassays, a spiked glucose solution, total human serum albumin, and glycated albumin were measured simultaneously. To test the performance of the developed sensor, clinical serum samples from healthy subjects and patients with diabetes were analyzed. The glucose level and glycation ratios of the clinical samples were determined with reasonable correlation. The R-squared values of glucose level and glycation ratio measurements were 0.932 and 0.930, respectively. The average detection recoveries of the sensor were 85.80% for glucose and 98.32% for the glycation ratio. The glucose level and glycation ratio in our results were crosschecked with reference diagnostic values of diabetes. Based on the outcomes of the present study, we propose that this novel platform can be utilized for the simultaneous detection of glucose and glycation ratios to diagnose and monitor diabetes mellitus.

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 μm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Paper spray ionization has been used as a fast sampling/ionization method for the direct mass spectrometric analysis of biological samples at ambient conditions. Here, we demonstrated that by utilizing paper spray ionization-mass spectrometry (PSI-MS) coupled with field asymmetric waveform ion mobility spectrometry (FAIMS), predictive metabolic and lipidomic profiles of routine breast core needle biopsies could be obtained effectively. By the combination of machine learning algorithms and pathological examination reports, we developed a classification model, which has an overall accuracy of 87.5% for an instantaneous differentiation between cancerous and noncancerous breast tissues utilizing metabolic and lipidomic profiles. Our results suggested that paper spray ionization-ion mobility spectrometry-mass spectrometry (PSI-IMS-MS) is a powerful approach for rapid breast cancer diagnosis based on altered metabolic and lipidomic profiles.

Many potential chemotherapeutics fail to reach patients. One of the key reasons is that compounds are tested during the drug discovery stage in two-dimensional (2D) cell cultures, which are often unable to accurately model in vivo outcomes. Three-dimensional (3D) in vitro tumor models are more predictive of chemotherapeutic effectiveness than 2D cultures, and thus, their implementation during the drug screening stage has the potential to more accurately evaluate compounds earlier, saving both time and money. Paper-based cultures (PBCs) are an emerging 3D culture platform in which cells suspended in Matrigel are seeded into paper scaffolds and cultured to generate a tissue-like environment. In this study, we demonstrate the potential of matrix-assisted laser desorption/ionization-mass spectrometry imaging with PBCs (MALDI-MSI-PBC) as a drug screening platform. This method discriminated regions of the PBCs with and without cells and/or drugs, indicating that coupling PBCs with MALDI-MSI has the potential to develop rapid, large-scale, and parallel mass spectrometric drug screens.

Chemiluminescence (CL) immunoassays for simultaneous detection of early acute myocardial infarction (AMI) biomarkers, including copeptin, heart-type fatty acid binding protein (h-FABP), and cardiac troponin I (cTnI), were developed by using Co2+/N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized magnetic carbon composite (Co2+-ABEI-Fe3O4@void@C) as an interface and a three-dimensional microfluidic paper-based device (3D μPAD) as a detection system. For CL immunoassays, Co2+-ABEI-Fe3O4@void@C was assembled with chitosan (CS) and gold nanoparticle-conjugated antibody (Au-Ab) sequentially to form the sensing platform (Co2+-ABEI-Fe3O4@void@C-CS/Au-Ab). In the presence of antigen (Ag), Ag was captured by the sensing interface to form an immunocomplex, leading to an increase in CL intensity due to the catalysis of -COO- existing in Ag. A 3D μPAD with three detection zones for simultaneous detection of copeptin, h-FABP, and cTnI was designed and fabricated to obtain time-resolved CL signals. Three kinds of immunocomplexes formed with copeptin, h-FABP, and cTnI were added to three detection zones of 3D μPAD, respectively. After injecting H2O2, three time-resolved CL signals were generated in one CL detection run by virtue of time-delayed transport of H2O2 to different detection zones. The three time-resolved CL signals were used for the simultaneous determination of copeptin, h-FABP, and cTnI. The detection limit of copeptin, h-FABP, and cTnI was 0.40 pg/mL, 0.32 pg/mL, and 0.50 pg/mL, respectively, which is at least 1 order of magnitude lower than most of the reported immunoassays. The immunoassays could be directly used for the detection of copeptin, h-FABP, and cTnI in human serum samples. The proposed immunoassays are simple, fast, sensitive, and selective, and are of great application potential in early diagnosis and treatment of AMI.

Multiclass chemical contamination of food has aroused ever-increasing attention due to the increasingly common findings of the co-occurrence of multiclass contamination, such as mycotoxin (aflatoxin M1, AFM1) and illegal additive (melamine, MEL). In the present study, a rapid, ultrasensitive detection paper was developed on the basis of a unique bridge-antibody label to realize on-site simultaneous detection of AFM1 and MEL in milk. This detection paper used the bridge-antibody label on fluorescent particles (i.e., the fluorescent Eu nanoparticles were first conjugated with polyclonal antibodies and then with monoclonal antibodies). Dramatically enhanced sensitivity was found, probably due to the increase in immobilization of efficient monoclonal antibodies onto microspheres. Under optimal conditions, the lower limits of detection were 0.009 and 0.024 ng/mL for AFM1 and MEL in milk, respectively, in comparison with similar works. Moreover, the cutoff values were 0.4 and 150 ng/mL for AFM1 and MEL, respectively. The recoveries ranged from 88.7% to 105.0% for AFM1 and from 84.6% to 117.7% for MEL, with relative standard deviations (RSDs) of 0.5-9.9% during the intraday and interday experiments. Comparison experiments conducted using the detection paper, HPLC, and UPLC-MS/MS found excellent agreement in the simultaneous detection of AFM1 and MEL in milk. This proposed method can be extensively employed for simultaneous monitoring of multiclass chemical contaminants to ensure food safety.

This paper focuses on one of the most commonly encountered materials in our society, namely paper. Paper is an inherently complex material, yet its use provides for chemical analysis approaches that are elegant in their simplicity of execution. In the first half of the previous century, paper in scientific research was used mainly for filtration and chromatographic separation. While its use decreased with the rise of modern elution chromatography, paper remains a versatile substrate for low-cost analytical tests. Recently, we have seen renewed interest to work with paper in (bio)analytical science, a result of the growing demand for inexpensive, portable analysis. Dried blood spotting, paper microfluidics, and paper spray ionization are areas in which paper is (re)establishing itself as an important material. These research areas all exploit several properties of paper, including stable sample storage, passive fluid movement and manipulation, chromatographic separation/extraction, modifiable surface and/or volume, easily altered shape, easy transport, and low cost. We propose that the real, and to date underexploited, potential of paper lies in utilizing its combined characteristics to add new dimensions to paper-based (bio)chemical analysis, expanding its applicability. This article provides the reader with a short historical perspective on the scientific use of paper and the developments that led to the establishment of the aforementioned research areas. We review important characteristics of paper and place them in a scientific context in this descriptive, yet critical, assessment of the achieved and the achievable in paper-based analysis. The ultimate goal is the exploration of integrative approaches at the interface between the different fields in which paper is or can be used.

Blood is the most intensely studied human biofluid and is used as the main indicator (so-called “gold standard”) of the health status of the individual. Blood is also a very complex fluid, containing a multiplicity of components, including cells and cell fragments (red and white blood cells, platelets), proteins, and many other organic molecules. Laboratory-based blood analysis is a standard procedure that provides a wealth of information about the concentration of key components and enables medical personnel to establish diagnoses and treatments. To date, options for point-of-care (POC) or point-of-use (POU) blood analysis are limited and very low-cost options even more so. One analytical platform that contains the promise to fill this need utilizes paper-based microfluidic devices. This is a research area that has exhibited significant growth in the past few years, primarily because of its simplicity, fundamental low cost, and versatility. This article reviews the basic properties of blood, how these pr...

The health risks associated with acute and prolonged exposure to estrogen receptor (ER) modulators has led to a concerted effort to identify and prioritize potential disruptors present in the environment. ER agonists and antagonists are identified with end-point assays, quantifying changes in cellular proliferation or gene transactivation in monolayers of estrogen receptor alpha expressing (ER+) cells upon exposure. While these monolayer cultures can be prepared, dosed, and analyzed in a highly parallelized manner, they are unable to predict the potencies of ER modulators in vivo accurately. Physiologically relevant model systems that better predict tissue- or organ-level responses are needed. To address this need, we describe here a screening platform capable of quantitatively assessing ER modulators in 96 chemically isolated 3D cultures. These cultures are supported in wax-patterned paper scaffolds whose design has improved performance and throughput over previously described paper-based setups. To highlight the potential of paper-based cultures for toxicity screens, we measured the potency of known ER modulators with a luciferase-based reporter assay. We also quantified the proliferation and invasion of two ER+ cell lines in the presence of estradiol. Despite the inability of the current setup to better predict in vivo potencies of ER modulators than monolayer cultures, the results demonstrate the potential of this platform to support increasingly complex and physiologically relevant tissue-like structures for environmental chemical risk assessment.

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detect...

In this work, we characterize the impact of large-volume processing upon the analytical sensitivity of flow-through paper-based immunoassays. Larger sample volumes feature greater molar quantities of available analyte, but the assay design principles which would enable the rapid collection of this dilute target are ill-defined. We developed a finite-element model to explore the operating conditions under which processing large sample volumes via pressure-driven convective flow would yield an improved binding signal. Our simulation results underscore the importance of establishing a high local concentration of the analyte-binding species within the porous substrate. This elevated abundance serves to enhance the binding kinetics, matching the time scale of target capture to the period during which the sample is in contact with the test zone (i.e., the effective residence time). These findings were experimentally validated using the rcSso7d-cellulose-binding domain (CBD) fusion construct, a bifunctional binding protein which adsorbs to cellulose in high abundance. As predicted by our modeling efforts, the local concentration achieved using the rcSso7d-CBD species is uniquely enabling for sensitivity enhancement through large-volume processing. The rapid analyte depletion which occurs at this high surface density also permits the processing of large sample volumes within practical time scales and flow regimes. Using these findings, we present guidance for the optimal means of processing large sample volumes for enhanced assay sensitivity.

This paper describes, for the first time, the use of oxidized buckypaper (BP) as a sorbent membrane of a stir-disc solid phase extraction module. The original device, consisting of a BP disc (d = 34 mm) enveloped in a polypropylene mesh pouch, was designed to extract organic micropollutants (OMPs) from environmental water samples in dynamic mode. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to analyze the extracts. Several classes of pesticides and pharmaceuticals were chosen as model compounds to evaluate key parameters affecting the recovery rates. To this end, the effects of adsorption time, desorption time, stirring speed, type and volume of solvent, and sample volume were thoroughly examined. After optimization, a novel and in-depth study was conducted to find a correlation between physicochemical properties of the analytes and extraction yields. Recoveries were mainly governed by a combination of logP and pKa values. As indicated, hydrophilic compounds with l...

Despite rapid progresses in single-cell analysis technologies, efforts to control the three-dimensional microenvironment for single cell measurements have been lacking. Here, we report a simple method to incorporate three-dimensional scaffolds, including polyvinylidene fluoride (PVDF) membranes and PVDF membrane replicated analog polydimethylsiloxane, into multiplexed single cell secretomic analysis platforms (including a microwell array and a single cell barcode microchip) to mimic the extracellular physical matrix and mechanical support for single cells. Applying this platform to brain tumor cell line U87 to investigate single cell protein secretion behavior on different substrates, we revealed that single cell protein secretions were regulated differently in three-dimensional (3D) microenvironments. This finding was further verified with intracellular cytokine staining, highlighting the significance of 3D single cell microenvironments. This new single cell biomimetic platform can be easily adaptable to other three-dimensional cell culture scaffolds or other single cell assays and may become a broadly applicable three-dimensional single cell analysis system to study the effect of microenvironment conditions on cellular functional heterogeneity in vitro.

Highly selective and sensitive detection of guanosine 3′-diphosphate-5′-diphosphate (ppGpp), namely, the stringent in plants or microorganisms responding to strict or extreme environmental conditions such as stress and starvation, which plays an important role in gene expression, rRNA and antibiotics production, regulations of virulence of bacteria, and growth of plants, faces a great challenge owing to its extreme similarity to normal nucleotides. By modifying the surface groups of a facile two-step hydrothermal route prepared carbon dots (CDs) with terbium ions (Tb3+) in this contribution, a novel fluorescent probe with excellent properties such as highly physical and chemical stability, narrow emission and excitation wavelength-independent emission was prepared. The Tb3+ ions on the surface of CDs cannot only preserve the intrinsic fluorescence (FL) of CDs but also keep its own coordination capacity with rare earth complex, and thus the clamp structure (four phosphate groups) of ppGpp can specific bind...

Paper-based cultures are an emerging platform for preparing 3D tissue-like structures. Chemical gradients can be imposed upon these cultures, generating microenvironments similar to those found in poorly vascularized tumors. There is increasing evidence that the tumor microenvironment is responsible for promoting drug resistance and increased invasiveness. Acidosis, or the acidification of the extracellular space, is particularly important in promoting these aggressive cancer phenotypes. To better understand how cells respond to acidosis there is a need for 3D culture platforms that not only model relevant disease states but also contain sensors capable of quantifying small molecules in the extracellular environment. In this work, we describe pH-sensing optodes that are capable of generating high spatial and temporal resolution maps of pH gradients in paper-based cultures. This sensor was fabricated by suspending microparticles containing pH-sensitive (fluorescein) and pH-insensitive (diphenylanthracene) dyes in a polyurethane hydrogel, which was then coated onto a transparent film. The pH-sensing films have a fast response time, are reversible, stable in long-term culture environments, have minimal photobleaching, and are not cytotoxic. These films have a pKa of 7.61 ± 0.04 and are sensitive in the pH range corresponding to normal and tumorigenic tissues. With these optodes, we measured the spatiotemporal evolution of pH gradients in paper-based tumor models.

This work reports a microfabricated electrochemical paper-based analytical device (ePAD) for the voltammetric determination of DNA. The device is patterned by wax-printing on paper and features a circular assay zone connected to an inlet zone and a sink via grooved microfluidic channels for accelerated flow rate. An electrochemical cell with integrated electrodes is formed on the reverse side of the paper by sputtering of thin metal films (Sn, Pt and Ag as the working, counter and reference electrode, respectively). Proof-of-principle of the ePAD for biosensing is demonstrated for a DNA assay involving attachment of capture DNA, hybridization with biotinylated target oligonucleotide and labeling with streptavidin-conjugated CdSe/ZnS quantum dots (QDs). After the acidic dissolution of the QDs, the released Cd(II) is quantified by anodic stripping voltammetry (ASV) at the Sn-film working electrode. Thanks to the synergistic effects of QDs amplification, the inherent sensitivity of ASV and the excellent detection capabilities of the Sn-film working electrode for Cd(II), the target DNA can be detected at levels as low as 0.11 pmol L

Porous media made of nitrocellulose and glass fiber are common "paper" substrates for lateral flow assays, microfluidic paper analytical devices and other point-of-care diagnostic assays. Such assays commonly use optical labels such as gold nanoparticles, latex beads, or fluorescent nanoparticles to visualize the presence of analytes. Fluorescent labels are commonly used in bioassays to enhance sensitivity, but autoluminescence of the paper substrate worsens signal-to-noise ratios of fluorescence-based assays. To date, there exists no systematic investigation of autoluminescence wavelengths or lifetimes of porous membranes used in lateral flow assays. In response, we quantified the autoluminescence of commonly used porous materials across the visible spectrum via excitation-emission spectroscopy and time-resolved fluorescence spectroscopy, and demonstrate that autoluminescence is solely due to autofluorescence with lifetimes of about 5 ns in the visible spectrum. Counterintuitively, we found that spectroscopy alone does not provide sufficient information to select candidate paper substrates for fluorophore-labeled assays. Therefore, we developed a simple quantitative framework to select a low-fluorescence substrate that minimizes both the overlap of paper and fluorophore emission spectra and the fluorescence intensity on an imaging system of interest (such as a gel imager). Use of this framework was shown to lower the limit of detection of an influenza A nucleoprotein immunoassay by over 50%. The tools developed in this manuscript enable assay developers to screen appropriate, low-fluorescence porous substrates and enhance the sensitivity of membrane-based fluorescence assays.

A pyrene based amphiphilic receptor has been utilized in the nanomolar detection of uric acid at physiological pH in water. The compound shows a unique concentration-dependent modulation in optical response toward uric acid. In intramolecular/static excimer form (low concentration range), it displays a ratiometric response, while a "turn-off" sensing is observed specifically in the presence of intermolecular/dynamic excimer (high concentration range). The present protocol is then employed for the estimation of uric acid in blood serum samples of healthy individuals. Bland-Altman analysis in comparison to clinically approved uricase assay indicated the high accuracy of the present method. Additionally, the extent of insect infestation in stored grain samples was determined by measuring the uric acid content of their aqueous extracts. Low-cost color strips were developed for on-site detection of uric acid without involving any sophisticated instrument or trained personnel.

The development of highly sensitive and selective methods for the detection of microRNA (miRNA) has attracted tremendous attention because of its importance in fundamental biological studies and diagnostic applications. In this work, we develop DNA-encoded Raman-active anisotropic nanoparticles modified origami paper analytical devices (oPADs) for rapid, highly sensitive, and specific miRNA detection. The Raman-active anisotropic nanoparticles were prepared using 10-mer oligo-A, -T, -C, and -G to mediate the growth of Ag cubic seeds into Ag nanoparticles (AgNPs) with different morphologies. The resulting AgNPs were further encoded with DNA probes to serve as effective surface-enhanced Raman scattering (SERS) probes. The analytical device was then fabricated on a single piece of SERS probes loaded paper-based substrate and assembled based on the principles of origami. The addition of the target analyte amplifies the Raman signals on DNA-encoded AgNPs through a target-dependent, sequence specific DNA hybridization assembly. This simple and low-cost analytical device is generic and applicable to a variety of miRNAs, allowing detection sensitivity down to 1 pM and assay time within 15 min, and therefore holds promising applications in point-of-care diagnostics.

Blood testing for endogenous small metabolites to determine physiological and biochemical states is routine for laboratory analysis. Here we demonstrate that by combining the commercial direct analysis in real time (DART) ion source with an ion trap mass spectrometer, native cholesterol in its free alcohol form is readily detected from a few hundred nanoliters of human serum loaded onto chromatography paper. Deuterium-labeled cholesterol was used as the internal standard to obtain the absolute quantity of the endogenous cholesterol. The amount of the cholesterol measured by this paper-loaded DART mass spectrometry (pDART-MS) is statistically comparable with that obtained by using commercially available fluorometric-enzymatic assay and liquid chromatography/mass spectrometry. Furthermore, sera from 21 participants at three different time points in an ultramarathon were collected to obtain their cholesterol levels. The test requires only very minimal sample preparation, and the concentrations of cholesterol in each sample were acquired within a minute.

We describe the design and characteristics of a paper-based analytical device for analyte concentration enrichment. The device, called a hybrid paper-based analytical device (hyPAD), uses faradaic electrochemistry to create an ion depletion zone (IDZ), and hence a local electric field, within a nitrocellulose flow channel. Charged analytes are concentrated near the IDZ when their electrophoretic and electroosmotic velocities balance. This process is called faradaic ion concentration polarization. The hyPAD is simple to construct and uses only low-cost materials. The hyPAD can be tuned for optimal performance by adjusting the applied voltage or changing the electrode design. Moreover, the throughput of hyPAD is 2 orders of magnitude higher than that of conventional, micron-scale microfluidic devices. The hyPAD is able to concentrate a range of analytes, including small molecules, DNA, proteins, and nanoparticles, in the range of 200−500-fold within 5 min.

The phosphorylation of nucleic acid with 5′-OH termini catalyzed by polynucleotide kinase (PNK) involves several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was first immobilized on the surface of aldehyde group modified paper, and BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL–1 and lysate of about 50 cells is achieved. The inhibition...

Current detection methods for paper-based analytical devices (PADs) rely on spectroscopic and electrochemical properties, which place special requirements on the analyte or need analyte labeling. Here, ion-transmission mass spectrometry (MS) was proposed for coupling with PADs to enable rapid in situ MS analysis of the sample on paper. The sample was analyzed directly on paper via analyte ionization by ions transmitted through the paper, generated by a low-temperature plasma probe. Prior to MS analysis, the sample can be separated by paper electrophoresis or by paper chromatography, among a variety of other features offered by PADs. The versatility of this technique was demonstrated by MS analysis of a paper microarray, a mixture of amino acids, and whole blood doped with drugs on PADs.

A novel label free spectrophotometric detection of malarial biomarker HRP-II following an indicator displacement assay has been developed. The assay is based on competitive displacement of murexide dye from its complex with Ni2+ by HRP-II present in serum samples. The binding constant (Kd) discerned for the dye and HRP-II to Ni2+ were 1.4 × 10–6 M–1 and 6.8 × 10–9 M–1, respectively. The progress of the reaction could be monitored from the change of color from orange (∼λ482 nm) to pink (∼λ515 nm) with the concomitant increase in HRP-II concentration in the mixture. A linear response (R2 = 0.995) curve was generated by plotting the ratio of absorbance (λ515 nm/λ482 nm) against the HRP-II concentrations. The method offers to detect HRP-II as low as 1 pM without any interference from some common salts and the major protein, HSA, present in the blood serum. The detection method was reproduced in a microfluidic paper based analytical device (μPAD), fabricated by printing hydrophobic alkyl ketene dimer on a chro...

Paper spray mass spectrometry has been demonstrated to be promising for direct analysis of therapeutic drugs in dried blood spots (DBS); however, the strong hydrogen bond and van de Waals interactions between paper substrate and analytes containing polar functional groups (e.g., therapeutic drugs) affect greatly the elution behavior and analysis sensitivity of compounds of interest during paper spray. Herein, we developed a one-sided ZrO2 coated paper substrate through a facile vacuum filtration approach using commercial ZrO2 particles as coating material and soluble starch as adhesive agent. Owing to the unique surface properties, as-prepared ZrO2 paper substrate has been shown to have excellent performance for analysis of therapeutic drugs in DBS during paper spray mass spectrometry. In contrast to original cellulose paper substrates, improvements of 43-189-fold in lower limit of quantitation (LLOQ) were obtained for the tested drugs using ZrO2 coated paper for paper spray. In comparing with the previously reported grade SG81 paper and one-sided silica coated paper, the LLOQs of the tested drugs with as-prepared ZrO2 paper decreased 1.5-16.5-fold relative to those from the above two, revealing that ZrO2 coated paper is a good candidate for paper spray in high sensitivity analysis of therapeutic drugs in DBS.

This paper describes the design and fabrication of a "pop-up" electrochemical paper-based analytical device (pop-up-EPAD) to measure beta-hydroxybutyrate (BHB)-a biomarker for diabetic ketoacidosis-using a commercial combination BHB/glucometer. Pop-up-EPADs are inspired by pop-up greeting cards and children's books. They are made from a single sheet of paper folded into a three-dimensional (3D) device that changes shape, and fluidic and electrical connectivity, by simply folding and unfolding the structure. The reconfigurable 3D structure makes it possible to change the fluidic path and to control timing; it also provides mechanical support for the folded and unfolded structures that enables good registration and repeatability on folding. A pop-up-EPAD designed to detect BHB shows performance comparable to commercially available plastic test strips over the clinically relevant range of BHB in blood when used with a commercial glucometer that integrates the ability to measure glucose and BHB (combination BHB/glucometer). With simple modifications of the electrode and the design of the fluidic path, the pop-up-EPAD also detects BHB in buffer using a simple glucometer-a device that is more available than the combination BHB/glucometer. Strategies that use a "3D pop-up"-that is, large-scale changes in 3D structure and fluidic paths-by folding/unfolding add functionality to EPADs (e.g., controlled timing, fluidic handling and path programming, control over complex sequences of steps, and alterations in electrical connectivity) and should enable the development of new classes of paper-based diagnostic devices.

In this work, a novel dual photoelectrochemical/colorimetric cyto-analysis format was first introduced into a microfluidic paper-based analytical device (μ-PAD) for synchronous sensitive and visual detection of H2O2 released from tumor cells based on an in situ hydroxyl radicals ((•)OH) cleaving DNA approach. The resulted μ-PAD offered an excellent platform for high-performance biosensing applications, which was constructed by a layer-by-layer modification of concanavalin A, graphene quantum dots (GQDs) labeled flower-like Au@Pd alloy nanoparticles (NPs) probe, and tumor cells on the surface of the vertically aligned bamboo like ZnO, which grows on a pyknotic Pt NPs modified paper working electrode (ZnO/Pt-PWE). It was the effective matching of energy levels between GQDs and ZnO levels that lead to the enhancement of the photocurrent response compared with the bare ZnO/Pt-PWE. After releasing H2O2, the DNA strand was cleaved by (•)OH generated under the synergistic catalysis of GQDs and Au@Pd alloy NPs and thus, reduced the photocurrent, resulting in a high sensitivity to H2O2 in aqueous solutions with a detection limit of 0.05 nmol observed, much lower than that in the previously reported method. The disengaged probe can result in catalytic chromogenic reaction of substrates, resulting in real-time imaging of H2O2 biological processes. Therefore, this work provided a truly low-cost, simple, and disposable μ-PAD for precise and visual detection of cellular H2O2, which had potential utility to cellular biology and pathophysiology.

In nucleic acid testing (NAT), gold nanoparticle (AuNP)-based lateral flow assays (LFAs) have received significant attention due to their cost-effectiveness, rapidity, and the ability to produce a simple colorimetric readout. However, the poor sensitivity of AuNP-based LFAs limits its widespread applications. Even though various efforts have been made to improve the assay sensitivity, most methods are inappropriate for integration into LFA for sample-to-answer NAT at the point-of-care (POC), usually due to the complicated fabrication processes or incompatible chemicals used. To address this, we propose a novel strategy of integrating a simple fluidic control strategy into LFA. The strategy involves incorporating a piece of paper-based shunt and a polydimethylsiloxane (PDMS) barrier to the strip to achieve optimum fluidic delays for LFA signal enhancement, resulting in 10-fold signal enhancement over unmodified LFA. The phenomena of fluidic delay were also evaluated by mathematical simulation, through which we found the movement of fluid throughout the shunt and the tortuosity effects in the presence of PDMS barrier, which significantly affect the detection sensitivity. To demonstrate the potential of integrating this strategy into a LFA with sample-in-answer-out capability, we further applied this strategy into our prototype sample-to-answer LFA to sensitively detect the Hepatitis B virus (HBV) in clinical blood samples. The proposed strategy offers great potential for highly sensitive detection of various targets for wide application in the near future.

We report the first portable upconversion nanoparticles (UCNPs)-based paper device for road-side field testing of cocaine. Upon the recognition of cocaine by two pieces of rationally designed aptamer fragments, the luminescence of UCNPs immobilized on the paper is quenched by Au nanoparticles (AuNPs), which indicates the cocaine concentration. This device can give quantitative results in a short time with high sensitivity using only a smartphone as the apparatus. Moreover, this device is applicable in human saliva samples, and it also can be used to monitor the cocaine content change in blood samples. The results of this work demonstrate the prospect of developing UCNPs-based paper devices for field testing of drug abuse.

A new sensor for the detection of hydrogen sulfide (H2S) gas has been developed to replace commercial lead(II) acetate-based test papers. The new sensor is a wet, porous, paper-like substrate coated with Bi(OH)3 or its alkaline derivatives at pH 11. In contrast to the neurotoxic lead(II) acetate, bismuth is used due to its nontoxic properties, as Bi(III) has been a reagent in medications such as Pepto-Bismol. The reaction between H2S gas and the current sensor produces a visible color change from white to yellow/brown, and the sensor responds to ≥ 30 ppb H2S in a total volume of 1.35 L of gas, a typical volume of human breath. The alkaline, wet coating helps the trapping of acidic H2S gas and its reaction with Bi(III) species, forming colored Bi2S3. The sensor is suitable for testing human bad breath and is at least 2 orders of magnitude more sensitive than a commercial H2S test paper based on Pb(II)(acetate)2. The small volume of 1.35-L H2S is important, as the commercial Pb(II)(acetate)2-based paper requires large volumes of 5 ppm H2S gas. The new sensor reported here is inexpensive, disposable, safe, and user-friendly. A simple, laboratory setup for generating small volumes of ppb-ppm of H2S gas is also reported.

Paper spray mass spectrometry is a method for the direct analysis of biofluid samples in which extraction of analytes from dried biofluid spots and electrospray ionization occur from the paper on which the dried sample is stored. We examined matrix effects in the analysis of small molecule drugs from urine, plasma, and whole blood. The general method was to spike stable isotope labeled analogs of each analyte into the spray solvent, while the analyte itself was in the dried biofluid. Intensity of the labeled analog is proportional to ionization efficiency, whereas the ratio of the analyte intensity to the labeled analog in the spray solvent is proportional to recovery. Ion suppression and recovery were found to be compound- and matrix-dependent. Highest levels of ion suppression were obtained for poor ionizers (e.g., analytes lacking basic aliphatic amine groups) in urine and approached -90%. Ion suppression was much lower or even absent for good ionizers (analytes with aliphatic amines) in dried blood spots. Recovery was generally highest in urine and lowest in blood. We also examined the effect of two experimental parameters on ion suppression and recovery: the spray solvent and the sample position (how far away from the paper tip the dried sample was spotted). Finally, the change in ion suppression and analyte elution as a function of time was examined by carrying out a paper spray analysis of dried plasma spots for 5 min by continually replenishing the spray solvent. Graphical Abstract ᅟ.

This paper addresses a growing need in clinical diagnostics for parallel, multiplex analysis of biomarkers from small biological samples. It describes a new procedure for assembling arrays of ssDNA and proteins on paper. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper and proceeds to assemble microzones of DNA-conjugated paper into arrays capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The synthesis of ssDNA oligonucleotides on paper is convenient and effective with 32% of the oligonucleotides cleaved and eluted from the paper substrate being full-length by HPLC for a 32-mer. These ssDNA arrays can be used to detect fluorophore-linked DNA oligonucleotides in solution, and as the basis for DNA-directed assembly of arrays of DNA-conjugated capture antibodies on paper, detect protein antigens by sandwich ELISAs. Paper-anchored ssDNA arrays with different sequences can be used to assemble paper-based devices capable of detecting DNA and antibodies in the same device and enable simple microfluidic paper-based devices.

We introduce a novel, facile, rapid, low-cost, highly reproducible, and power-free synthesizable fabrication method of paper-based silver nanoparticle (AgNP) immersed surface-enhanced Raman scattering (SERS) platform, known as the successive ionic layer absorption and reaction (SILAR) method. The rough and porous properties of the paper led to direct synthesis of AgNPs on the surface as well as in the paper due to capillary effects, resulting in improved plasmon coupling with interparticles and interlayers. The proposed SERS platform showed an enhancement factor of 1.1 × 10(9), high reproducibility (relative standard deviation of 4.2%), and 10(-12) M rhodamine B highly sensitive detection limit by optimizing the SILAR conditions including the concentration of the reactive solution (20/20 mM/mM AgNO3/NaBH4) and the number of SILAR cycles (six). The applicability of the SERS platform was evaluated using two samples including human cervical fluid for clinical diagnosis of human papillomavirus (HPV) infection, associated with cervical cancer, and a malachite green (MG) solution for fungicide and parasiticide in aquaculture, associated with human carcinogenesis. The AgNP-immersed SERS-functionalized platform using the SILAR technique allowed for high chemical structure sensitivity without additional tagging or chemical modification, making it a good alternative for early clinical diagnosis of HPV infection and detection of MG-activated human carcinogenesis.

A water-soluble glucopyranosyl conjugate, L, has been synthesized and characterized by different analytical and spectral techniques. The L has been demonstrated to have switch-on fluorescence enhancement of ∼75 fold in the presence of La(3+) among the nine lanthanide ions studied in the HEPES buffer at pH 7.4. A minimum detection limit of 140 nM (16 ± 2 ppb) was shown by L for La(3+) in the buffer at physiological pH. The utility of L has been demonstrated by showing its sensitivity toward La(3+) on Whatman filter paper strips. The reversible and reusable action of L has been demonstrated by monitoring the fluorescence changes as a function of the addition of La(3+) followed by F(-) and HPO4(2-) ions. The complexation of L by La(3+) was shown by absorption spectra wherein isosbestic behavior was observed. The Job's plot suggests a 2:1 complex between L and La(3+), and the same was supported by ESI-MS. The control molecular study revealed the necessity of hydroxy quinoline and the amine group for La(3+) ion binding and the glyco-moiety to bring water solubility and biocompatibility. The structural features of the [2L+La(3+)] complex were established by DFT computational calculations. The chemo-ensemble, [2L+La(3+)], is shown responsible for providing intracellular fluorescence imaging in HepG2 cells.

In this study, a paper-based point-of-care (POC) colorimetric biosensor was developed for the detection of lactate dehydrogenase in serum using a nonporous, oxygen impermeable reversibly gelling polysaccharide material based on pullulan. The pullulan could be printed onto paper surfaces along with all required assay reagents, providing a means for high-stability immobilization of all reagents on paper. Serum containing lactate dehydrogenase (LDH) was directly spotted on to the pullulan-coated bioactive paper and provided quantitative colorimetric data that was comparable to that obtained with a conventional plate-reader method. The paper strip was found to be highly stable and could be stored at 4 °C for at least 10 weeks with no loss in performance, as compared to a complete loss in performance within 1 day when the reagents were printed without the stabilizing polysaccharide. The ease of fabrication coupled with the high stability of the printed reagents provides a facile platform for easily manufactured POC sensors.

On-line coupling of electrochemistry (EC) to mass spectrometry (MS) is a powerful approach for identifying intermediates and products of EC reactions in situ. In addition, EC transformations have been used to increase ionization efficiency and derivatize analytes prior to MS, improving sensitivity and chemical specificity. Recently, there has been significant interest in developing paper-based electroanalytical devices as they offer convenience, low cost, versatility, and simplicity. This report describes the development of tubular and planar paper-based electrochemical cells (P-EC) coupled to sonic spray ionization (SSI) mass spectrometry (P-EC/SSI-MS). The EC cells are composed of paper sandwiched between two mesh stainless steel electrodes. Analytes and reagents can be added directly to the paper substrate along with electrolyte, or delivered via the SSI microdroplet spray. The EC cells are decoupled from the SSI source, allowing independent control of electrical and chemical parameters. We utilized P-EC/SSI-MS to characterize various EC reactions such as oxidations of cysteine, dopamine, polycyclic aromatic hydrocarbons, and diphenyl sulfide. Our results show that P-EC/SSI-MS has the ability to increase ionization efficiency, to perform online EC transformations, and to capture intermediates of EC reactions with a response time on the order of hundreds of milliseconds. The short response time allowed detection of a deprotonated diphenyl sulfide intermediate, which experimentally confirms a previously proposed mechanism for EC oxidation of diphenyl sulfide to pseudodimer sulfonium ion. This report introduces paper-based EC/MS via development of two device configurations (tubular and planar electrodes), as well as discusses the capabilities, performance, and limitations of the technique.

This paper demonstrates that, for applications in resource-limited environments, expensive microplate spectrophotometers that are used in many central laboratories for parallel measurement of absorbance of samples can be replaced by photometers based on inexpensive and ubiquitous, consumer electronic devices (e.g., scanners and cell-phone cameras). Two devices, (i) a flatbed scanner operating in transmittance mode and (ii) a camera-based photometer (constructed from a cell phone camera, a planar light source, and a cardboard box), demonstrate the concept. These devices illuminate samples in microtiter plates from one side and use the RGB-based imaging sensors of the scanner/camera to measure the light transmitted to the other side. The broadband absorbance of samples (RGB-resolved absorbance) can be calculated using the RGB color values of only three pixels per microwell. Rigorous theoretical analysis establishes a well-defined relationship between the absorbance spectrum of a sample and its corresponding RGB-resolved absorbance. The linearity and precision of measurements performed with these low-cost photometers on different dyes, which absorb across the range of the visible spectrum, and chromogenic products of assays (e.g., enzymatic, ELISA) demonstrate that these low-cost photometers can be used reliably in a broad range of chemical and biochemical analyses. The ability to perform accurate measurements of absorbance on liquid samples, in parallel and at low cost, would enable testing, typically reserved for well-equipped clinics and laboratories, to be performed in circumstances where resources and expertise are limited.

Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.

The analytical performance and a suggested mechanism for zero volt paper spray using chromatography paper are presented. A spray is generated by the action of the pneumatic force of the mass spectrometer (MS) vacuum at the inlet. Positive and negative ion signals are observed, and comparisons are made with standard kV paper spray (PS) ionization and nanoelectrospray ionization (nESI). While the range of analytes to which zero volt PS is applicable is very similar to kV PS and nESI, differences in the mass spectra of mixtures are interpreted in terms of the more significant effects of analyte surface activity in the gentler zero volt experiment than in the other methods due to the significantly lower charge. The signal intensity of zero volt PS is also lower than in the other methods. A Monte Carlo simulation based on statistical fluctuation of positive and negative ions in solution has been implemented to explain the production of ions from initially uncharged droplets. Uncharged droplets first break up due to aerodynamics forces until they are in the 2-4 ?m size range and then undergo Coulombic fission. A model involving statistical charge fluctuations in both phases predicts detection limits similar to those observed experimentally and explains the effects of binary mixture components on relative ionization efficiencies. The proposed mechanism may also play a role in ionization by other voltage-free methods. (Figure Presented). � 2015 American Chemical Society.

An inexpensive, disposable and highly selective microfluidic paper-based analytical device (μPAD) is described for the determination of ammonia (molecular ammonia and ammonium cation) in wastewaters which implements for the first time a gas-diffusion separation step on a paper-based platform. Its hydrophilic reagent zones were defined by printing filter paper with a hydrophobic paper sizing agent using a conventional inkjet printer. The sample was introduced into the sodium hydroxide impregnated sample zone of the μPAD. This allowed the quantitative conversion of the ammonium ion to molecular ammonia which diffused across the hydrophobic microporous Teflon membrane of the device into an adjacent hydrophilic reagent zone containing the acid-base indicator 3-nitrophenol or bromothymol blue. The change in indicator color was measured using a desktop scanner for ammonia quantification. Under optimal conditions, the μPAD is characterized by a limit of detection of 0.8 and 1.8 mg N L(-1) and repeatability of 3.1 and 3.7% (n ≥ 10, 20 mg N L(-1)), expressed as relative standard deviation, in the case of 3-nitrophenol or bromothymol blue, respectively. This μPAD was used successfully for the determination of ammonia in sewage and soil water samples. The small dimensions, minimal reagent consumption, low cost, simplicity of operation, and possibility of using a portable scanner make the proposed μPAD suitable for on-site ammonia monitoring in contaminated environmental waters and domestic, agricultural and industrial wastewaters. The successful implementation of the gas-diffusion approach on a paper-based platform is expected to result in the development of other μPADs for volatile analytes.

A versatile point-of-care assay platform was developed for simultaneous detection of multiple targets based on a microfluidic paper-based analytic device (μPAD) using a target-responsive hydrogel to mediate fluidic flow and signal readout. An aptamer-cross-linked hydrogel was used as a target-responsive flow regulator in the μPAD. In the absence of a target, the hydrogel is formed in the flow channel, stopping the flow in the μPAD and preventing the colored indicator from traveling to the final observation spot, thus yielding a "signal off" readout. In contrast, in the presence of a target, no hydrogel is formed because of the preferential interaction of target and aptamer. This allows free fluidic flow in the μPAD, carrying the indicator to the observation spot and producing a "signal on" readout. The device is inexpensive to fabricate, easy to use, and disposable after detection. Testing results can be obtained within 6 min by the naked eye via a simple loading operation without the need for any auxiliary equipment. Multiple targets, including cocaine, adenosine, and Pb(2+), can be detected simultaneously, even in complex biological matrices such as urine. The reported method offers simple, low cost, rapid, user-friendly, point-of-care testing, which will be useful in many applications.

We report the first demonstration of using heat on a paper device to rapidly concentrate a clinically relevant analyte of interest from a biological fluid. Our technology relies on the application of localized heat to a paper strip to evaporate off hundreds of microliters of liquid to concentrate the target analyte. This method can be used to enrich for a target analyte that is present at low concentrations within a biological fluid to enhance the sensitivity of downstream detection methods. We demonstrate our method by concentrating the tuberculosis-specific glycolipid, lipoarabinomannan (LAM), a promising urinary biomarker for the detection and diagnosis of tuberculosis. We show that the heat does not compromise the subsequent immunodetectability of LAM, and in 20 min, the tuberculosis biomarker was concentrated by nearly 20-fold in simulated urine. Our method requires only 500 mW of power, and sample flow is self-driven via capillary action. As such, our technology can be readily integrated into portable, battery-powered, instrument-free diagnostic devices intended for use in low-resource settings.

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL(-1) (10(2) pmol L(-1)), a value within the range of clinically relevant concentrations.