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A novel microfluidic paper-based analytical device (μPAD) was developed with benzoquinone (BQ)-mediated E. coli respiration method to measure the biotoxicities of pollutants. Functional units including sample injection, fluid-cell separation, all-carbon electrode-enabled electrochemical detection, were integrated on a piece of chromatography paper. The three-electrode, working electrode, counter electrode and reference electrode, were simultaneously screen-printed on the μPAD with conductive carbon ink. The satisfying electrochemical performance of the paper-based carbon three-electrode was confirmed by cyclic voltammetry detecting K3 [Fe(CN)6]. The process of cell toxication was considered that toxicants inhibited cell respiration and diminished the electrons on E. coli respiratory chain. It was quantitatively reflected by measuring oxidation current of hydroquinone (HQ) as a reduced state of the redox mediator BQ after the incubation of cells with pollutants. The current detection time, BQ concentration and E. coli incubation time were carefully optimized to establish the systematic optimized operations of BQ-mediated E. coli respiration method. Using the fabricated μPAD the half inhibitory concentration (IC50) were Cu2+ solution 13.5 μg mL−1, Cu2+-soil 21.4 mg kg−1, penicillin sodium-soil 85.1 mg kg−1, and IC30 of Pb2+ solution was 60.0 μg mL−1. Detection of pesticide residues in vegetable juices were accomplished in a similar way. The proposed method is fascinating on three points; 1) The generality in the biotoxicity detection depends on toxicants inducing cellular respiratory inhibition; 2) The portability and affordability make it convenient for practical applications, because of replacing incubators and centrifuges; 3) There is potential applicability in less-developed areas due to its simple operation and low-cost.

Food safety issues, especially those related to drug residues, have caused concern in recent years. Malachite green (MG), which is frequently used as ectoparasiticide, fungicide, and antiseptic in fish farming, is poisonous and carcinogenic. Several technologies and methods have been developed to accurately detect MG, but they can be time-consuming and expensive. Herein, we prepared a glass fiber paper as a surface-enhanced Raman scattering substrate for rapid and convenient detection of MG residues in fish. Uniform silver nanoparticles were deposited on the glass fiber paper by two in situ reduction steps of silver nitrate. The Raman signal of MG was detected and recorded using a portable Raman spectrometer in 10 s. There was a good linear relationship between the surface-enhanced Raman scattering signal intensity and the concentration of MG between 1 × 10−7 and 1 × 10−5 mol/L and the limit of detection was 5 × 10−10 mol/L. Residual MG in fish samples was extracted and detected with a recovery rate range of 98.9–119.4%. This method provides a novel way to detect MG in fish.

Paper based assays are paving the way to automated, simplified, robust and cost-effective point of care testing (POCT). We propose a method for fabricating three dimensional (3D) microfluidic paper based analytical devices (μPADs) via combining thin adhesive films and paper folding, which avoids the use of cellulose powders and the complex folding sequence and simultaneously permits assays in several layers. To demonstrate the effectiveness of this approach, a 3DμPADs was designed to conduct more assays on a small footprint, allowing dual colorimetric and electrochemical detections. More importantly, we further developed a 3D platform for implementing automated and multiplexed ELISA in parallel, since ELISA, a routine and standard laboratory method, has rarely been used in practical analyses outside of the laboratory. In this configuration, complex and multistep diagnostic assays can be carried out with the addition of the sample and buffer in a simple fashion. Using Troponin I as model, the device showed a broad dynamic range of detection with a detection limit of 0.35 ng/mL. Thus, the developed platforms allow for various assays to be cost-effectively carried out on a single 3D device, showing great potential in an academic setting and point of care testing under resource-poor conditions.

Microfluidic paper-based analytical devices (μPADs) for detection of hydrogen peroxide and glucose have been developed. The analytical performance of colorimetric detection using the conventional starch-iodine color reaction has been significantly improved by using gelatin as the surface modifier which retains the enzyme activity in the dry filter paper strip, improves antioxidability, as well as decreases the strong background signal. Under optimal conditions, the color intensities show a good linear relationship with glucose concentration ranging from 0.5 to 5 mM and hydrogen peroxide concentration from 0.5 to 6 mM, with the detection limit of 0.05 mM and 0.1 mM, respectively. In addition, the accuracy of colorimetric sensor has been successfully assessed in detecting glucose from real human serum samples and recovery value ranges from 95.7% to 97%, which are approaching to the glucose oxidase endpoint. The new colorimetric assay exhibits high sensitivity, good selectivity, acceptable stability and reproducibility. The present approach is promising for monitoring glucose for point of care diagnostic applications, especially in regions with resource-limited settings.

Paper-based biosensor is one of the most commonly used platforms for point-of-care testing (POCT). Among these platforms, microfluidic paper-based analytical devices (μPADs) have the most versatile designs due to the different hydrophobic barrier patterns and layers of the devices. In addition, μPADs can also be used in combination with other biosensor platforms to improve the performance of the device. Simple and convenient methods for fabricating low-cost and design-adjustable hydrophobic barriers on paper are one of the most challenging aspects for creating μPADs. This work demonstrated a simple technique for using the common polylactic acid (PLA) filament and wax filament to create hydrophobic barriers on paper for μPADs using a commercialized 3D printer. As a proof of concept, the papers with 3D printed PLA barrier were used in combination with a fluidic chip in a prototype biosensor, in which the barrier paper housed four cell-free reactions and the fluidic chip achieved sample delivery to the reactions in the device. Our designed prototype was capable of discriminating dengue virus serotypes based on small nucleotide sequence differences. The proposed combination of 3D-printed barrier paper and fluidic chip provides a versatile platform for rapid prototyping of POCT with possible compatibility with various detection systems.

Norfloxacin (NOR) in milk may influence mammalian cell replication and bring about a decrease in the efficiency for treating infection in humans. However, current techniques for detecting NOR usually require expensive instruments and trained personnel. In this work, we have developed a low-cost and simple method via paper-based fluorescent immunoassay for highly sensitive and selective detection of NOR in milk at picogram level. The NOR monoclonal antibody labeled with quantum dots is used as a detection probe to recognize the corresponding NOR, which can quantitatively detect NOR on paper-based devices. The detection limits in aqueous solution and milk are 1 pg/mL and 10 pg/mL, respectively. The developed paper-based method provides a cheap, sensitive, eco-friendly, and rapid approach for quantitative detection of trace NOR in milk, which may find wide applications in food safety inspection. Noteworthy, the method is especially suitable for applications at resource-limited and on-site settings.

We report the colorimetric detection of dopamine (DA) on microfluidic paper-based analytical devices (μPADs) using an oxidation-reduction method. Here, dopamine reacts with ferric chloride forming reduced Fe2+ that subsequently reacts with phenanthroline to form the red tris(1,10-phenanthroline)iron(II) complex. The devices were fabricated by wax printing and changes in color intensity were recorded using a common cell phone. Subsequent analysis using Photoshop software, yielded a limit of detection (LOD) for DA of 0.37 μmol/L with a linear range of 0.527–4.75 μmol/L and relative standard deviation of 0.11% (inter-day) and 0.15% (intra-day) for n = 15 paper chips. The effects of detection conditions have been investigated and are discussed. Cow serum samples and human blood serum and plasma samples were detected. The work, herein, demonstrates the potential of this method as a low cost and rapid colorimetric technique to detect DA in real samples.

Microfluidic paper-based analytical devices (μPADs) have recently emerged as a simple, portable, user-friendly, and affordable alternative to more instrument-intensive analytical approaches for point-of-care testing (POCT), food safety analysis, and environmental monitoring. However, most of the existing methods for the fabrication of μPADs still face a great challenge because of different trade-offs among cost, convenience, and the pattern resolution. In this work, we report a facile one-step approach to prepare a μPAD using an affordable, easy-to-build 3D printer to generate patterns of solid wax on laboratory filter paper. The presented wax printing method did not require the use of predesigned masks and an external heat source to form complete hydrophobic wax barrier through the use of a custom-made extruder. The results revealed a strong linear relationship (R2 = 0.985) between the nominal and the printed widths of the wax barriers. The achievable resolution of the wax barrier printed on filter paper was 468 ± 72 µm, which was lower than previously reported minimum barrier feature sizes achieved by wax printing and other wax patterning techniques, such as stamping and screen-printing. The analytical utility of the fabricated μPADs was evaluated for colorimetric nitrite and glucose detection in artificial solutions. It was found that the fabricated μPADs provided adequate accuracy and reproducibility for quantitative determination of nitrite and glucose within concentration ranges relevant to the disease detection in human saliva and urine. The wax printing approach reported here provides a simple, rapid, and cost-effective fabrication method for paper-based microfluidics and may bring benefits to medical diagnostics in the developing world.

The analysis of exosomes, which shows an increasing potential as prognostic biomarkers for non-invasive cancer diagnosis, can reveal their biological functions and disease associations. Nevertheless, the development of a convenient and quantitative method for determination of exosomes is still challenging. Herein, a novel approach for exosome quantification using pH test paper is developed via HRP-mediated promotion of mussel-inspired surface engineering and reagent-free functionalization of urease molecules. Uerase can hydrolyse urea into ammonia and carbon dioxide, and simultaneously raise the pH value of the solution. By establishing the relationship between exosome recognition and the change of pH value of the sensing solution, we can directly employ the low-cost, widely used and commercially available pH test paper to quantitatively analyse exosomes. The pH-responsive bioassay enables sensitive detection of exosomes with a detection of limit down to 4.46 × 103 particles/µL and can be successfully applied for determination of exosomes in clinical specimens. The versatility and reliability of our sensing platform may open up opportunities towards paper-based diagnosis of cancer.

Under controlled dispersion conditions, sample injection towards a detector opened essential fields for the Analytical Chemistry fast development methods. Flow injection analysis (FIA) and batch injection analysis (BIA) systems are crucial for injecting the sample in these analytical methods. The BIA system eliminated the flow manifold, with samples injected directly onto the detector inside the batch injection cell. Paper was slightly evaluated coupled to FIA, and no reports were found associated with BIA. Still, it can potentially reduce the BIA manifold by removing the batch injection cell based on the capillarity properties to disperse the injected solution over the detection system. Hence, this article reported the first work coupling batch-injection analysis and microfluidic paper-based analytical device (BIA-μPAD) with pencil-drawn electrodes directly attached to the paper using a CO

In this work, we analyzed the performance of the Electro-Filtering Paper Spray Ionization (EFSI) method for detecting compounds in unprocessed samples. A relatively rigid and electrically conductive copper filter was used as a substrate to insure sufficient and efficient sample-solvent extraction and to increase the conductivity for paper spraying. The method was demonstrated as applicable for indirect high-throughput analysis of large-volume unprocessed samples, which is not possible with conventional nanoESI or direct paper spray methods. The new method can generate different desired ion signals for a wide range of compounds by selecting different extraction solvents. Moreover, key parameters related to extraction efficiency were optimized in detail to obtain the most satisfactory extraction efficiency during antibiotic analysis. Finally, under optimal conditions, the EFSI method was successfully used to detect four antibiotics in animal products of egg, chicken, and chicken liver, exhibiting good reproducibility with calibration curves between 81.6% and 96.3%, and R2 values above 0.99. Recoveries of 75.0%ￚ94.6% were obtained for the four antibiotics. Hence, the proposed EFSI-MS is a successful, economical, rapid, and high-throughput method that is effective for both unknown and targeted extraction of unprocessed samples by mass spectrometric analysis.

Nowadays, environmentally friendly synthesis pathways for preserving the environment and minimizing waste are strongly required. Herein, we propose filter paper as a convenient scaffold for chemical reactions. To demonstrate this novel approach, Prussian Blue Nanoparticles (PBNPs) were synthesized on filter paper by utilizing few μL of its precursors without external inputs, i.e. pH, voltage, reducing agents, and without producing waste as well. The functional paper, named “Paper Blue”, is successfully applied in the sensing field, exploiting the reduction of hydrogen peroxide at low applied potential. The eco-designed “Paper Blue” was combined with wax- and screen-printing to manufacture a reagentless electrochemical point-of-care device for diabetes self-monitoring, by using glucose oxidase as the biological recognition element. Blood glucose was linearly detected for a wide concentration range up to 25 mM (450 mg/dL), demonstrating its suitability for management of diabetes and glucose-related diseases. The Paper Blue-based biosensor demonstrated a correlation coefficient of 0.987 with commercial glucose strips (Bayer Contour XT). The achieved results demonstrated the effectiveness of this approach, which is also extendible to other (bio)systems to be applied in catalysis, remediation, and diagnostics.

Several solution-based methods have recently been adapted for use in paper substrates for enzymatic amplification to increase the number of copies of DNA sequences. There is limited information available about the impact of a paper matrix on DNA amplification by enzymatic processes, and about how to optimize conditions to maximize yields. The work reported herein provides insights about the impact of physicochemical properties of a paper matrix, using nuclease-assisted amplification by exonuclease III and nicking endonuclease Nt. Bbv, and a quantum dot (QD) - based Forster Resonance Energy Transfer (FRET) assay to monitor the extent of amplification. The influence of several properties of paper on amplification efficiency and kinetics were investigated, such as surface adsorption of reactants, and pore size. Additional factors that impact amplification processes such as target length and the packing density of oligonucleotide probes on the nanoparticle surfaces were also studied. The work provides guidance for development of more efficient enzymatic target-recycling DNA amplification methods in paper substrates.

In modern times, viruses still threaten people's lives. Among them, norovirus was the main pathogenic factor in the cause of gastroenteritis and foodborne illness, of which the GII.4 and GII.17 genotypes are prevalent in China and most parts of the world. A simple and low-cost platform for rapid and accurate norovirus detection remains a major challenge. After the cell-free system and paper-based chromogenic system were optimized, a rapid and specific norovirus detection method was established based on norovirus-specific sequences in combination with toehold switch elements. The development of a visible color change during detection eliminates the need for any complicated instruments. We validated this strategy and its specificity in differentiating GII.4, GII.17, Zika virus, and human coronavirus HKU1. The results showed that the optimized detection system not only provided a simple and rapid detection method for the sufficient differentiation of the two norovirus genotypes but also showed high specificity and no cross-reactivity with other viruses. Using nucleic acid isothermal amplification, this assay showed a limit of detection of 0.5 pM for the GII.4 genotype and 2.6 fM for the GII.17 genotype in reactions that could be observed directly with the naked eye. Our results suggested that this paper-based colorimetric method could serve as a simple and low-cost visual detection method for pathogens in clinical samples, especially in remote or rural areas.

Herein, a simple microfluidic paper-based analytical device (μPAD) by using platinum nanoparticles (Pt NPs) as highly active peroxidase mimic for simultaneous determination of glucose and uric acid was fabricated. The μPAD consisted of one sample transportation layer, four paper-based detection chips, and two layers of hydrophobic polyethylene terephthalate (PET) films. The four detection chips were immobilized with various chromogenic reagents, Pt NPs, and specific oxidase (glucose oxidase or uricase). H2O2 generated by specific enzymatic reactions could oxidize co-immobilized chromogenic reagents to produce colored products by using Pt NPs as efficient catalyst. The multi-layered structure of μPAD could effectively improve the color uniformity and color intensity. Total color intensity from each two detection chips modified with distinct chromogenic reagents were used for quantitative analysis of glucose and uric acid, respectively, resulting in significantly improved sensitivity. The linear range for glucose and uric acid detection was 0.01–5.0 mM and 0.01–2.5 mM, respectively. Satisfied results were obtained for glucose and uric acid detection in real serum samples. An easy-to-use smartphone APP was developed for convenient and intelligent detection. The developed μPAD integrated with smartphone as detector holds great applicability for simple and portable on-site analysis.

Paper-based analytical devices (PADs) with four new designs could be fabricated using commercially available home-based scan-and-cut printer. They serve for miniaturised platforms for chemical analysis. Replication analysis of a sample together with the calibration (using the analyte standards at different concentrations) can be completed in a single run, by utilising smartphone as the detector. Some new approaches for choosing detection zones were suggested. The four proposed PAD designs here were used as models in microliter scale operation to demonstrate the well-known chemistries of colorimetric determinations of iron, phosphate, and hardness using 1,10-phenanthroline and simple aqueous guava leaf extract; molybdate, and EBT-EDTA complexometric titration, respectively, through calibrations: where Blue (B) value = 88.2log [Fe3+] - 80.8, R2 = 0.989; B value = 1.75 [Fe3+] + 0.198, R2 = 0.999; Grey scale (I) value = 1.77 [Fe3+] – 1.22, R2 = 0.997; Red (R) value = 16.1log [PO43−] + 8.95, R2 = 0.999; Hue (H) value = 43.3log [Ca2+] + 233, R2 = 0.994, respectively. For the hardness, using one of the PAD designs, true titration was also possible. Applications of the proposed devices and procedures were demonstrated for real world samples with validation. Additionally, kinetic study of the molybdenum blue for phosphate was demonstrated using one of the PADs.

The microfluidic paper-based analytical devices (μPADs) have grown-up swiftly over the decade due to its low cost, simple fabrication procedure, resource-limitedness, non-toxicity and their environmentally benign nature. The μPADs, also identified as point-of-care devices or health care devices have successfully applied in several fields such as diagnostics, biological, food safety, environmental, electrochemical and most importantly colorimetric/fluorimetric sensors, owing to the attractive passive motions of analyte without any external forces. In recent years, a large number of colorimetric and fluorimetric probes have been reported that can selectively recognize the analytes in μPADs. However, there is no organized review on its structure-activity relationship. In this review, we have focused to summarize the colorimetric and fluorimetric probes utilized in μPADs. This review discuss about the relationships between the structure and functions of various probes as signaling units of the efficient μPADs. The probes including nanomaterials, nanozymes, polymers and organic molecules, their structural activity with regard to sensing performances along with their limit of detection are also discussed. This review is expected to assist readers for better understanding of the sensing mechanisms of various chemo and bio-probes utilized in μPADs, as well as promote their advancement in the field. On the other hand, this review also helps the researchers for enhancement of μPADs and paves way for synergistic application of existing molecular probes as an effective diagnostic tool for the worldwide pandemic novel corona virus COVID-19.

In this work, two different microfluidic paper-based analytical devices (μPADs) were developed for the quantification of nitrite and nitrate in human saliva samples, in order to aid in the diagnosis of some diseases and health conditions associated with these ions. The development of these nitrite and nitrate μPADs involved several studies to optimize their design and construction, including an interference assessment and stability studies. These μPADs allowed a nitrite determination in a range of 5–250 μM with limits of detection and quantification of 0.05 μM and 0.17 μM, respectively, and a nitrate determination in the range 0.2–1.2 mM with limits of detection and quantification of 0.08 mM and 0.27 mM, respectively. As for the stability, both of the μPADs were stable when stored in vacuum at 4 °C (the nitrite μPAD for at least 60 days and the nitrate μPAD for at least of 14 days) and, after the sample placement, the nitrite and nitrate μPADs could be scanned within the first 4 and 2 h, respectively. The nitrite μPAD measurements were compared with the ones obtained from the standard colorimetric method and there were no statistically significant differences between these two methods. To evaluate the accuracy of nitrate μPAD measurements, 4 certified water samples were used and recovery studies using saliva samples were performed.

MicroRNAs (miRNAs) are considered as the potential biomarkers for many cancers. To determine miRNAs in cancer cells is significant for realizing these diseases. In this work, a microfluidic paper-based laser-induced fluorescence sensor based on duplex-specific nuclease (DSN) amplification was developed and applied to selectively and sensitively determine miRNAs in cancer cells. An interface for laser-induced fluorescence detection was firstly applied to perform the sample detection on the paper-based chip. Under the optimal conditions, DSN (3 μL 0.10 U) and Taqman probes (2 μL 2.5 × 10−7 M) were preserved on the circles (Diameter 4 mm) of the folded paper chip. When miRNA solution was added, the mixed solution could trigger fluorescence signal amplification by cyclically digesting hybrids of miRNAs and Taqman probes by DSN. The whole determination, including sample heating process, could be accomplished within 40 min. The detection limits for miRNA-21 and miRNA-31 were 0.20 and 0.50 fM respectively, corresponding to only 1.0 and 1.5 zmol consumption of miRNAs. The testing of mismatched miRNAs showed that the method had good specificity. Finally, the method was applied to determine miRNA-21 and miRNA-31 in lysates of cancer cells of A549 and HeLa, and hepatocyte LO2. MiRNA-21 and miRNA-31 could be successfully found from the two cancer cells. The concentrations for miRNA-21 and miRNA-31 were 1.74 × 10−13 M and 6.29 × 10−14 M in HeLa cell lysate (3.75 × 104 cells/mL), 3.07 × 10−15 M and 3.28 × 10−15 M in A549 cell lysate (8.33 × 106 cells/mL) respectively. The recoveries ranged from 87.30% to 111.83%, indicating the results were reliable. The developed method was effective, selective and sensitive in the determination of miRNAs in cancer cells.

Vitamin B

Photoacoustic (PA) effect has been widely applied in many fields, e.g., physics, chemistry and biomedicine. Herein, a miniaturized PA device is developed by integrating laser source, photo chopper, PA cell, microphone, and laptop for point-of-care testing in bioassay. With glucose assay as model, a piece of paper strip preloading chitosan, starch-potassium iodide (KI) and glucose oxidase (GOD) as lab-on-paper is employed for loading sample prior to PA detection. In the presence of glucose, the product generated on the paper strip would give rise to a strong PA signal in the PA cell under the irradiation of frequency-modulated laser at 520 nm via laptop readout. With a sample volume of 20 μL, a detection limit of 0.03 mM is obtained for glucose assay, along with a linear range of 0.08–1 mM. The accuracy and practicability of the present PA device is well demonstrated by detecting glucose in whole blood. Differing from the conventional PA instrument, the present PA device is really small in bulk with competitive sensitivity and excellent stability, offering a promising tool for point-of-care testing in bioassay.

Rapid, on-site analysis was achieved through significantly simplified operation procedures for a wide variety of toy samples (crayon, temporary tattoo sticker, finger paint, modeling clay, and bubble solution) using a miniature mass spectrometry system with ambient ionization capability. The labor-intensive analytical protocols involving sample workup and chemical separation, traditionally required for MS-based analysis, were replaced by direct sampling analysis using ambient ionization methods. A Mini β ion trap miniature mass spectrometer was coupled with versatile ambient ionization methods, e.g. paper spray, extraction spray and slug-flow microextraction nanoESI for direct identification of prohibited colorants, carcinogenic primary aromatic amines, allergenic fragrances, preservatives and plasticizers from raw toy samples. The use of paper substrates coated with Co3O4 nanoparticles allowed a great increase in sensitivity for paper spray. Limits of detection as low as 5μgkg-1 were obtained for target analytes. The methods being developed based on the integration of ambient ionization with miniature mass spectrometer represent alternatives to current in-lab MS analysis operation, and would enable fast, outside-the-lab screening of toy products to ensure children's safety and health.

A novel recognition element of molecularly imprinted films (MIFs) was synthesized by wet phase inversion (WPI) on the surface of Ti/TiO2 electrode for highly selective and sensitive electrochemical detection of bisphenol A (BPA). The Ti/TiO2/MIFs sensor was constructed by casting the precursor poly(acrylonitrile-co-acrylic acid) (p(AN-co-AA)) in dimethyl sulfoxide containing template molecule BPA onto the electrode and then immersing into water, resulting in simultaneous p(AN-co-AA) precipitation and BPA imprinting via the facile WPI. The imprinted sites could selectively rebind BPA through hydrogen bonding and hence lead to the equalizing current increase in amperometric detection, by which the BPA could be sensed electrochemically. Accordingly, the Ti/TiO2/MIFs sensor offered a favorable linearity within the wide range over five orders of magnitude (4.4nM-0.13mM), and a low detection limit down to 1.3nM. Excellent recognition selectivity for BPA was also attained over its analogues. Furthermore, this sensor was successfully applied to detect BPA in seawater and paper cup samples, and high recoveries were 86-110% with low relative standard deviations of 1.3-3.2%. By using BPA as a model, the MIFs-based method may provide a facile, rapid, and cost-effective way for ultrasensitive electrochemical measurements of various targeted compounds with good applicability to WPI.

A novel simple and readily synthesized turn-on fluorescent probe 4-(benzo[d]thiazol-2-yl)-1,3-phenylene bis(2-chloroacetate) (BPBC) for cysteine (Cys) was reported. This probe was designed based on an excited-state intramolecular proton transfer (ESIPT) dye: benzothiazole, and two chloroacetate groups present in benzothiazole as the reaction sites for Cys. It shows high selectivity and sensitivity for Cys over other amino acids including the similar structured homocysteine (Hcy) and glutathione (GSH). In addition, probe BPBC was successfully applied to bioimage intracellular Cys in living cells with low cytotoxicity. More importantly, a paper test strip system was developed with probe BPBC for Cys detection conveniently.

In this work, ZnO nanorods (ZnO NRs) with different sizes were hydrothermally grown on the surface of Whatman filter paper for the fabrication of a microfluidic paper-based device (μPAD) for the simultaneous detection of glucose and uric acid. As dual enzymatic reaction was employed for the colorimetric detection in this μPAD, the presence of ZnO NRs promoted the enzyme immobilization thus significantly enhancing the colorimetric signal. The coffee ring effect was effectively conquered by the uniform distribution of ZnO NR as well as a specialized double-layered μPAD design. Meanwhile, two color indicators with distinct colors were used to provide complementary results to better quantify the concentration of the analytes by naked eye. As a result, two linear calibration curves were obtained for the detection of glucose (0.01–10 mmol L−1) and uric acid (0.01–5 mmol L−1), along with a LOD of 3 μmol L−1 for glucose and 4 μmol L−1 for uric acid, respectively. The practical usefulness of the proposed μPAD was further validated by the simultaneous analysis of glucose and uric acid in serum samples and urine samples.

The detection of individual chromophores that contribute to the overall discoloration of paper or pulp ("yellowing") is a challenge because these substances are only present in extremely small amounts (ppm to ppb range). In this work, paper spray (PS) coupled with mass spectrometry was used to detect a low-concentrated cellulosic key-chromophore, 2,5-dihydroxyacetophenone (DHAP). Sensitivity was enhanced by derivatization with Girard's reagent T (GT). DHAP was successfully detected in historic paper samples and also was applied to model papers in order to investigate different factors that influence its generation: temperature, time, relative humidity, and the presence of iron ions, by means of a full factorial design. The main factors, temperature and relative humidity, have the most impact on the generation of DHAP, but the interactions between the factors are also significant and are therefore important for the degradation process. The historical papers containing DHAP were then compared to the artificially aged samples. The results were confirmed by independent, accurate mass measurements.

Paper with immobilized protein can provide a low-cost platform for diagnostics, but evidence is emerging that the instability of protein on paper during shipping and storage severely limits its commercial development. Of these, paper-immobilized antibody, though widely used, is in urgent need of improvement in the shelf life. Herein we provided a detailed investigation of a simple and versatile approach for achieving stable antibody on paper to improve bioassay performance. Anti-Blood Group A IgM was chosen as the model antibody and its activity was tested via agglutination reactions. Representative polysaccharides (i.e., chitosan, sodium alginate and dextran) were used as protectants and their ability to stabilize antibody under heat and desiccation stresses was investigated. It was found that the presence of dextran with high concentration markedly stabilized paper-immobilized IgM for at least 120 days. Our results indicate that dextran with good film-forming ability could be a protectant for paper-immobilized antibody.

A new sensitive non-invasive gold nanoparticle-based sensor that enables to detect thiols in the human skin has been developed. The detection procedure implied the assessment of the color change of a paper sensor resulting from aggregation of gold nanoparticles caused by thiols. The ratio of the intensity of the photo image blue channel vs the red one (in units of RGB coloration) served as analytical response. The main thiol in the skin is glutathione, therefore, it was used as model biothiol and spiking substance. The range of linearity for glutathione was 8–75 µM, the detection limit was 6.9 µM. RSD≤7% is for inter-day determination of 10 μM glutathione and RSD≤12% is the intra-day value. The recovery of 5 µM and 10 µM of glutathione was evaluated by applying solution, containing thiol-spikes, on skin. The results varied in the range 77–138%. A hundred-fold excess of serine, alanine, histidine, threonine, creatinine, urea, and ammonia; a ten-fold excess of glycine, proline, leucine, isoleucine, phenylalanine, asparagine; and a five-fold excess of valine, tryptophan, tyrosine, and uric acid, which can be extracted from the skin and is contained in the test matrix, have no significant effect on 10 µM glutathione signal. Thiols level in the skin of volunteers (21–65 years old, men and women) detected with the use of a proposed non-invasive sensor was 11.6–47.5 µM.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic haemolytic disorder. Most persons with G6PD deficiency are asymptomatic, but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce haemolysis, which is commonly found in Asian countries. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis. This study proposes a novel quantitative method to detect G6PD deficiency using paper-based analytical devices (G6PDD-PAD). Wax printing was utilized for fabricating circular reaction zone patterns in paper. The colorimetric assay is based on the formation of formazan via a reduction of tetra-nitro blue tetrazolium (TNBT) by the G6PD enzyme on G6PDD-PAD. Detection was achieved by capturing the colour using a desktop scanner and the colour intensity was analysed with Adobe Photoshop C56. The results showed that the G6PD activity analysed by G6PDD-PAD was highly correlated with the standard biochemical assay (SBA) (r2=0.87, p

Acute myocardial infarction (AMI) causes significant mortality and morbidity. The determination of multiple AMI biomarkers is very important for the timely diagnosis of AMI. In this work, simultaneous determination of three AMI biomarkers were achieved by virtue of a three-dimensional (3D) microfluidic paper-analytical device (μPAD) with temporally resolved chemiluminescence (CL) emissions for the first time. A dual-signal amplification strategy was introduced including by employing primary antibody functionalized gold nanoparticles (Ab1-GNPs) immobilized on the detection zone as amplified capture probes, and Co(II) catalyst, secondary antibody, luminol multifunctionalized gold nanoparticles (Co(II)-Ab2-luminol-GNPs) with excellent CL activity as amplified signal probes. CL immunoreactions were performed at three detection zone of the fabricated 3D μPAD by assembling Ab1-GNPs, antigen, and Co(II)-Ab2-luminol-GNPs to form sandwich-type immunocomplexes. Auto separated CL signals with temporal resolution were obtained by time delayed transport of H2O2 to different detection zones for multiplexed analysis. The CL signal obtained by using Co(II)-Ab2-luminol-GNPs as signal probe (10576 a.u.) were about 20-fold higher than that by using conventional horseradish peroxidase labeled antibody modified luminol-GNPs as signal probe (531 a.u.). Finally, three AMI biomarkers including heart-type fatty acid-binding protein (H-FABP), cardiac troponin I (cTnI) and copeptin were quantitatively analyzed in one CL detection run by reading the CL intensity of the obtained three CL emission peaks. The detection range were ultra-wide ranged from 0.1 pg/mL to 1 μg/mL, 0.5 pg/mL to 1 μg/mL and 1 pg/mL to 1 mg/mL with the detection limits down to 0.06 pg/mL, 0.3 pg/mL and 0.4 pg/mL for H-FABP, cTnI and copeptin detection, respectively. The developed μPAD based immunoassay performing multiplexed analysis ability, high sensitivity, ultra-wide dynamic range, favorable selectivity, accessible accuracy and reproducibility, have great application potential for the early diagnosis of AMI.

A dual functional molecularly imprinted polymer (MIP) coated paper (MIP-CP) sensor was firstly proposed for rapid fluorescent (FL) and visual analysis of norfloxacin (NOR) in aqueous solutions. The sensor was fabricated via a sol-gel technique, and characterized by FTIR and SEM. The extraction capacity, selectivity, detection limit, and linear range of the MIP-CP sensor were studied by an FL spectrophotometer. The modified paper sensor showed good selectivity and high extraction capacity for NOR and its structural analogues. For FL analysis of NOR, the linear range and detection limit were found to be 10–100 ng/ml and 2.59 ng/ml, respectively. The visual determination of NOR (under UV light) based on the MIP-CP sensor was also explored, and the determination limit was found to be 0.1 μg/ml. Finally, the proposed methods were successfully applied to the direct determination of NOR in pharmaceutical preparations and cross-validated by UPLC. The MIP-CP sensor might be a potential platform for antibiotic determination due to its multifunction and good analytical performance.