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Microbially contaminated food can cause serious health hazards and economic losses, therefore sensitive, rapid, and highly specific visual detection is called for. Traditional detection of microorganisms is complex and time-consuming, which cannot meet current testing demands. The emergence of paper-based biosensors provided an effective method for efficient and visual detection of microorganisms, due to its high speed, all-in-one device, low cost, and convenience. This review focused on 5 biomarkers, namely nucleic acids, proteins, lipopolysaccharides, metabolites, and the whole microorganism of microorganisms. Besides, the recognition methods were summed up in 5 forms, including immunological recognition, aptamer recognition, nucleic acid amplification-mediated recognition, DNAzyme recognition and clustered regularly interspaced short palindromic repeats mediated recognition. In addition, we summarized the applications of paper-based biosensors in the detection of microorganisms thoroughly. Through the exploration of different biomarkers, identification methods, and applications, we hope to provide a reference for the development of paper-based biosensors and their application in safeguarding the food chain.

A paper-based electrochemical dual-function biosensor capable of determining pH and TCO2 was synthesized for the first time using an iridium oxide pH electrode and an all-solid-state ion electrode (ASIE). In the study, to obtain highly reliable results, the biosensor was equipped with a real-time pH correction function before TCO2 measurements. Compared to traditional liquid-filling carbon dioxide detection sensors, the utilization of ferrocene endows our novel sensor with abundant positive sites, and thus greatly improves its performance. Conversely, the introduction of MXene with conductivity close to that of metals reduces electrode resistance, which is beneficial for accelerating the electrochemical reaction of the sensor and reducing LOD. After optimization, the detection range of TCO2 is 0.095 nM–0.66 M, with a detection limit of as low as 0.023 nM. In addition, the sensor was used in real serum sample-spiked recovery experiments and comparison experiments with existing clinical blood gas analyzers, which confirmed the effectiveness of its clinical application. This study provides a method for the rational design of paper-based electrochemical biosensors and a new approach for the clinical detection of blood carbon dioxide. [ABSTRACT FROM AUTHOR]

Paper-based biosensors are a potential paradigm of sensitivity achieved via microporous spreading/microfluidics, simplicity, and affordability. In this paper, we develop decorated paper with graphene and conductive polymer (herein referred to as graphene conductive polymer paper-based sensor or GCPPS) for sensitive detection of biomolecules. Planetary mixing resulted in uniformly dispersed graphene and conductive polymer ink, which was applied to laser-cut Whatman filter paper substrates. Scanning electron microscopy and Raman spectroscopy showed strong attachment of conductive polymer-functionalized graphene to cellulose fibers. The GCPPS detected dopamine and cytokines, such as tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) in the ranges of 12.5–400 µM, 0.005–50 ng/mL, and 2 pg/mL–2 µg/mL, respectively, using a minute sample volume of 2 µL. The electrodes showed lower detection limits (LODs) of 3.4 µM, 5.97 pg/mL, and 9.55 pg/mL for dopamine, TNF-α, and IL-6 respectively, which are promising for rapid and easy analysis for biomarkers detection. Additionally, these paper-based biosensors were highly selective (no serpin A1 detection with IL-6 antibody) and were able to detect IL-6 antigen in human serum with high sensitivity and hence, the portable, adaptable, point-of-care, quick, minute sample requirement offered by our fabricated biosensor is advantageous to healthcare applications. [ABSTRACT FROM AUTHOR]

Sepsis is an immune response to a microbial invasion that causes organ injury and dysfunction due to a systemic inflammatory response. Sepsis is a serious, life-threatening condition and a widely recognized global health challenge. Given its high death rate, it is critical to diagnose sepsis and start treatment as early as possible. There is an urgent need for a sensitive and rapid screening method for detecting sepsis. In this study, we investigated the use of MMP-9 as a biomarker for sepsis. A colorimetric paper-based biosensor was used for the detection of MMP-9 utilizing peptide-magnetic nanoparticle conjugates. The method is based on the cleavage of the MMP-9-specific peptide by the protease leading to the detaching of the magnetic beads from the sensor surface and changing of color. A fecal intraperitoneal (FIP) challenge was used to induce sepsis in mice, and an MMP-9 secretion was measured by taking blood and Bronchoalveolar Lavage (BAL) fluid samples at 1 h, 2 h, 4 h, and 20 h (early sepsis) post-challenge intervals. The results of the paper-based sensor for the detection of MMP-9 levels in blood samples and BAL samples were compared with ELISA and Western Blot. We found that both blood and BAL levels of MMP-9 increased immediately and could be detected as early as 1 h in FIP mice post-challenge. Our work adds evidence to the assertion that MMP-9 is a reliable biomarker for the detection of sepsis at early stages. [ABSTRACT FROM AUTHOR]

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Micro RNAs (miRNAs) are 19–23 nucleotides-long non-coding RNA which have been identified as important biomarkers for many diseases, including cancers. Here, we report an ultrasensitive, highly specific, label-free, and without nucleic acid amplification (NAA) detection of miRNAs using paper-based single-walled carbon nanotubes (SWNTs) field-effect transistor (FET) biosensors. The strategy involved a two-step protocol starting with direct hybridization of the target miRNA with a specific RNA probe immobilized on SWNTs networks deposited on the paper substrate to generate the first response, followed by the recognition of the resulting RNA/miRNA duplexes with the Carnation Italian ringspot virus p19 protein (p19) in a size-dependent manner, giving the second electrical response that magnified the biosensor sensitivity. As a demonstration, we detected the miRNA-122a, a promising biomarker for the diagnosis of early-stage hepatocellular carcinoma (HCC) in various sample matrices, including phosphate buffers, human serum, and synthetic saliva. We achieved the lowest detection of 0.1 aM and a wide dynamic range of 0.1 aM–1 fM, that demonstrated its potential for rapid, facile, low-cost, and point-of-care detection of miRNAs for early cancer diagnosis.

As the current pandemic has shown, lateral flow assays (LFAs) are a prime example of point-of-care devices enabling quick testing at an affordable price. However, their ease of use undeniably affects their sensitivity, making them less sensitive than other multi-step and time-consuming diagnostic assays, such as polymerase chain reactions and enzyme-linked immunosorbent assays. A possible solution to overcome this lack of sensitivity is the exploitation of bottom-up approaches to synthesize nanomaterials with outstanding properties for use as colorimetric labels in LFAs, that is, using nanoparticles with better optical capabilities to improve the generation of the colorimetric signal and the overall sensitivity of LFAs. Following this strategy, we rationally optimized the synthesis of gold and iridium oxide nanoflowers (Au–IrO2 NFs) to enhance their physical–chemical properties as colorimetric labels in LFAs. Specifically, we were able to rationally control their size (from 155 to 53 nm in diameter) in order to guarantee an optimal flow along the different pads of a LFA. Moreover, thanks to their superior plasmonic behavior (compared to standard AuNPs), we could achieve an 8.5-fold lower limit of detection (down to 1.2 ng/mL) for human immunoglobulin G (HIgG) than standard LFAs (10.1 ng/mL). Therefore, due to their optical and redox properties, bioconjugation capabilities, and synergic combination of the individual components, Au–IrO2 NFs appear as potential candidates for the next generation of optical LFAs. [ABSTRACT FROM AUTHOR]

Paper-based analytical devices are promising options for lab-on-chip and other biodetection applications. Among various nanomaterials, carbon quantum dots (CQDs) are famous for their significant and stable photoluminescence (PL) properties, as well as their fast and cost-effective gram-scale synthesis techniques. However, studies on the durability and sustainability of their performance as fluorescent composites are very limited and not systematic. Therefore in this investigation, the PL emission intensity of cellulose papers containing various types of CQDs, including pure, N-, S-, P-, N/S-, and N/P-doped CQDs, were studied over two months, at three different storage temperatures, namely, 4 °C, 10 °C, and 25 °C, and four pH settings as to be 5, 6, 7 and 8. The results show that the environmental keeping conditions, as well as the chemical structure of the embedded CQDs, have significant impacts on the durability and sustainability of the produced sensors. All the fluorescent composites investigated in this study proved to have very high performances, sustaining from above 60–90% of their PL emission intensities over time. Although 4 °C is proved to be generally the best keeping temperature for all the fluorescent nanocomposites, the best pH, in which the PL is most sustained, depends strongly on the chemical nature of the embedded CQDs. The CQDs/cellulose composites were kept for additional longer durations, above six months, in their best keeping conditions, and their performance was checked, showing the reliability of the proposed setup for further industrial promising applications. [ABSTRACT FROM AUTHOR]

An electrochemical paper-based sensor was developed for the detection of bacterial infection (BI)-specific biomarker procalcitonin (PCT). Reduced graphene oxide-gold nanoparticles (rGO-AuNP) and poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) were synthesized and were fabricated to a disposable, portable, and inexpensive cellulose fiber paper (CFP) substrate. rGO-AuNP-PEDOT:PSS nanocomposite-modified conductive paper-based biosensing platform was efficaciously fabricated by a constant and simple coating procedure. rGO-AuNP-PEDOT:PSS nanocomposite-modified conductive paper electrode was found to provide a sensitive and conductive substrate for PCT detection. The presence of rGO-AuNP-PEDOT:PSS nanocomposite on CFP substate was investigated by Fourier transform infrared spectrometry, field emission scanning electron microscopy, ultraviolet–visible spectroscopy, and X-ray diffraction studies. The electrochemical behavior of rGO-AuNP-PEDOT:PSS @CFP surface was studied with impedance spectroscopy, cyclic voltammetry, and chronoamperometry techniques. This low-cost paper-based biosensor has a linear range for PCT of 1 × 103 to 6 × 107 fg mL−1. This developed sensor exhibited good reproducibility with a relative standard deviation (RSD) of about 3.7%. The proposed CFP-based biosensor has been proven as an accelerated simple point-of-care (POC) exploratory approach for early PCT diagnosis in inadequate areas with limited production facilities, computational techniques, and highly skilled experts. [ABSTRACT FROM AUTHOR]

Diagnostic blood tests can guide the administration of healthcare to save and improve lives. Most clinical biosensing blood tests require a trained technician and specialized equipment to process samples and interpret results, which greatly limits test accessibility. Colorimetric paper-based diagnostics have an equipment-free readout, but raw blood obscures a colorimetric response which has motivated diverse efforts to develop blood sample processing techniques. This work uses inexpensive readily-available materials to engineer user-friendly dilution and filtration methods for blood sample collection and processing to enable a proof-of-concept colorimetric biosensor that is responsive to glutamine in 50 µL blood drop samples in less than 30 min. Paper-based user-friendly blood sample collection and processing combined with CFPS biosensing technology represents important progress towards the development of at-home biosensors that could be broadly applicable to personalized healthcare. [ABSTRACT FROM AUTHOR]

Paper-based biosensors are a potential paradigm of sensitivity achieved via microporous spreading/microfluidics, simplicity, and affordability. In this paper, we develop decorated paper with graphene and conductive polymer (herein referred to as graphene conductive polymer paper-based sensor or GCPPS) for sensitive detection of biomolecules. Planetary mixing resulted in uniformly dispersed graphene and conductive polymer ink, which was applied to laser-cut Whatman filter paper substrates. Scanning electron microscopy and Raman spectroscopy showed strong attachment of conductive polymer-functionalized graphene to cellulose fibers. The GCPPS detected dopamine and cytokines, such as tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6) in the ranges of 12.5–400 µM, 0.005–50 ng/mL, and 2 pg/mL–2 µg/mL, respectively, using a minute sample volume of 2 µL. The electrodes showed lower detection limits (LODs) of 3.4 µM, 5.97 pg/mL, and 9.55 pg/mL for dopamine, TNF-α, and IL-6 respectively, which are promising for rapid and easy analysis for biomarkers detection. Additionally, these paper-based biosensors were highly selective (no serpin A1 detection with IL-6 antibody) and were able to detect IL-6 antigen in human serum with high sensitivity and hence, the portable, adaptable, point-of-care, quick, minute sample requirement offered by our fabricated biosensor is advantageous to healthcare applications.

Point-of-care testing (POCT) biosensors are rapidly developing in recent years because of the urgent needs for rapid test and home test. Among various types of POCT biosensors, paper-based biosensors have occupied large market share owing to the advantages of cost-effectiveness, portability and simplicity. To further meet the increasing requirement of POCT in various application scenes, the functionalization of paper is highly demanded. Metal-organic framework (MOF), a multifunctional porous material, is introduced in the design of paper-based biosensors, significantly improving the sensing property and application potential. This review provides a summary of the recent advances and emerging trends of MOF-functionalized paper-based biosensors (MOF@paper), including different types of substrates, fabrication methods, detection mechanisms and applications. On account of the multifunctionality and superior performance of MOF@paper biosensors, this field should possess promising prospect in scientific researches, translational medicine and clinical applications. • Multifunctional MOF endows paper-based biosensors with diverse detection properties. • The substrates and fabrication methods for MOF@paper biosensors are reviewed. • Various detection mechanisms are summarized and discussed. • MOF@paper biosensors are promising in diagnosis, food, environment and other related applications. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one of the most common primary tumors in the liver. Diagnosis of HCC was investigated by detecting Glypican-3 (GPC3) protein using two approaches based on nanotechnology: developing a biosensor using a screen-printed electrode and designing and fabricating a paper-based biosensor using paper chromatography. The developed approaches were modified with multi-walled carbon nanotubes. Next, immobilized with anti-Glypican-3 antibodies, then deposited by native cow Bovine Serum Albumin (BSA). The characterizing techniques included scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier-transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). Moreover, differential pulse voltammetry (DPV) was used to detect GPC3 protein in a concentration range of 0.0001 ng/ml to 100 ng/ml. In this study, screen-printed electrodes achieved high sensitivity (i.e., the limit of detection (LOD) equals 0.495 pg/ml) of GPC3 along with correlation coefficient (R2) equals 0.996. Meanwhile, paper-based electrodes attained slightly lower sensitivity (LOD of 0.597 pg/ml) and R2 of 0.983. [ABSTRACT FROM AUTHOR]

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

Due to the common contamination of multiple mycotoxins in food, which results in stronger toxicity, it is particularly important to simultaneously test for various mycotoxins for the protection of human health. In this study, a disposable immunosensor array with low-cost was designed and fabricated using cellulose paper, polydimethylsiloxane (PDMS), and semiconducting single-walled carbon nanotubes (s-SWCNTs), which was modified with specific antibodies for mycotoxins AFB1 and FB1 detection. The strategy for fabricating the immunosensor array with two individual channels involved a two-step protocol starting with the form of two kinds of carbon films by depositing single-wall carbon nanotubes (SWCNTs) and s-SWCNTs on the cellulose paper as the conductive wire and sensing element, followed by the assembly of chemiresistive biosensor with SWCNTs strip as the wire and s-SWCNTs as the sensing element. After immobilizing AFB1-bovine serum albumin (AFB1-BSA) and FB1-bovine serum albumin (FB1-BSA) separately on the different sensing regions, the formation of mycotoxin-BSA-antibody immunocomplexes transfers to electrochemical signal, which would change with the different concentrations of free mycotoxins. Under optimal conditions, the immunosensor array achieved a limit of detection (LOD) of 0.46 pg/mL for AFB1 and 0.34 pg/mL for FB1 within a wide dynamic range from 1 pg/mL to 20 ng/mL. Furthermore, the AFB1 and FB1 spiked in the ground corn and wheat extracts were detected with satisfactory recoveries, demonstrating the excellent practicality of this established method for simultaneous detection of mycotoxins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Diagnostic blood tests can guide the administration of healthcare to save and improve lives. Most clinical biosensing blood tests require a trained technician and specialized equipment to process samples and interpret results, which greatly limits test accessibility. Colorimetric paper-based diagnostics have an equipment-free readout, but raw blood obscures a colorimetric response which has motivated diverse efforts to develop blood sample processing techniques. This work uses inexpensive readily-available materials to engineer user-friendly dilution and filtration methods for blood sample collection and processing to enable a proof-of-concept colorimetric biosensor that is responsive to glutamine in 50 µL blood drop samples in less than 30 min. Paper-based user-friendly blood sample collection and processing combined with CFPS biosensing technology represents important progress towards the development of at-home biosensors that could be broadly applicable to personalized healthcare.

Foodborne illnesses caused by Escherichia coli (E. coli) are causing worldwide morbidity and mortality, threating human health. One way to limit and eliminate E. coli infections is developing fast and sensitive methods for detection. Biosensors have emerged to complement traditional strategies for E. coli detection. This review aims at providing an updated summary of developed biosensors for specific detection of E. coli in the last 5 years. Since biosensor development first initiated with immobilizing biorecognition element on the transducer, this review starts with a brief discussion on the immobilization methods of biorecognition elements on the biosensor surface, followed by a detailed discussion focused on the progresses made in principles and applications of various biorecognition element-based biosensors for E. coli detection. Especially, the limitations of each method using various biorecognition elements are discussed. Furthermore, the advancements of paper-based biosensors are generalized. In addition, a few suggestions and challenges for prospective development of biosensor are also provided. [ABSTRACT FROM AUTHOR]

The demand for sensitive, portable, and non-destructive analysis of pathogenic bacteria is of significance in point-of-care diagnosis. Herein, we constructed a smart electrochemiluminescence (ECL) biosensor by integrating a flexible paper-based sensing device and a disposable three-electrode detecting system. Staphylococcus aureus (S. aureus)-responsive cellulose paper was prepared by employing aptamer as recognition element and a probe DNA (probe DNA-GOD) tagged with glucose oxidase (GOD) as a signal amplification unit. The formation of aptamer- S. aureus complex mediated the quantitative release of probe DNA-GOD. The remaining probe DNA-GOD on the paper-based aptasensor was then activated by glucose, which resulted in a significant decrease in ECL signal. To further improve the ECL performance of biosensor, a large number of Ru(bpy) 3 2+ molecules were embedded into porous zinc-based metal-organic frameworks (MOFs) to form Ru(bpy) 3 2+ functionalized MOF nanoflowers (Ru-MOF-5 NFs). Such biosensor enabled accurate, non-destructive, and real-time monitoring of S. aureus -contaminated food samples, opening a new avenue for sensitive recognition of pathogenic bacteria. [Display omitted] • A wearable, disposable and smart paper-based biosensor was developed. • The sensor realized the sensitive, in situ and non-destructive analysis of S. aureus. • The limit of detection from our sensor was better than that of some existing methods. • The practical application in the field of S. aureus -contaminated foods was feasible. [ABSTRACT FROM AUTHOR]

To enhance the sensitivity of lateral flow assays (LFAs), a simple strategy is proposed using a nitrocellulose membrane modified with a superabsorbent polymer (SAP). SAP was incorporated into a nitrocellulose membrane for the flow control of detection probes. When absorbing aqueous solutions, SAP promoted the formation of biomolecule complexes to achieve up to a tenfold sensitivity improvement for the detection of human IgG. The assay time was optimized experimentally and numerically to within 20 min using this strategy. Moreover, fluid saturation in LFAs modified with SAP was mathematically simulated to better understand the underlying process, and molecular dynamics simulations were carried out to determine the effect of SAP. The proposed design was also applied to samples spiked with human immunoglobulin-depleted serum to test its applicability. The strategy presented is unique in that it preserves the characteristics of conventional LFAs, as it minimizes user intervention and is simple to manufacture at scale. [ABSTRACT FROM AUTHOR]

Due to their unique properties, paper-based biosensors have attracted attention as inexpensive devices for on-site analysis. To achieve fast and sensitive detection of analytes, immobilization of enzymes with high apparent activities on paper is highly desirable; however, this is challenging. Herein, we report an improved approach to attach a malathion degrading enzyme, PoOPHM9, on paper via an interlocking network of Pluronic F127 (PF127)–poly(acrylic acid)–enzyme conjugates. The addition of PF127 improved retention of enzymatic activity as the apparent kinetic constant Vmax of the immobilized enzyme increased two-fold compared with the paper prepared without PF127. The PF127–poly(acrylic acid)–PoOPHM9 papers provided rapid colorimetric detection of malathion at 0.1–50 mM. The detection was completed within 5 min using a smartphone and image analysis software. As a proof-of-concept, malathion-contaminated water, plant, and apple samples were analyzed with the papers successfully. This material is promising for on-site rapid analysis of malathion-contaminated samples. [ABSTRACT FROM AUTHOR]

A paper-based electrochemical dual-function biosensor capable of determining pH and TCO 2 was synthesized for the first time using an iridium oxide pH electrode and an all-solid-state ion electrode (ASIE). In the study, to obtain highly reliable results, the biosensor was equipped with a real-time pH correction function before TCO 2 measurements. Compared to traditional liquid-filling carbon dioxide detection sensors, the utilization of ferrocene endows our novel sensor with abundant positive sites, and thus greatly improves its performance. Conversely, the introduction of MXene with conductivity close to that of metals reduces electrode resistance, which is beneficial for accelerating the electrochemical reaction of the sensor and reducing LOD. After optimization, the detection range of TCO 2 is 0.095 nM-0.66 M, with a detection limit of as low as 0.023 nM. In addition, the sensor was used in real serum sample-spiked recovery experiments and comparison experiments with existing clinical blood gas analyzers, which confirmed the effectiveness of its clinical application. This study provides a method for the rational design of paper-based electrochemical biosensors and a new approach for the clinical detection of blood carbon dioxide., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Paper-based biosensors are considered simple and cost-efficient sensing platforms for analytical tests and diagnostics. Here, a paper-based electrochemical biosensor was developed for the rapid and sensitive detection of microRNAs (miRNA-155 and miRNA-21) related to early diagnosis of lung cancer. Hydrophobic barriers to creating electrode areas were manufactured by wax printing, whereas a three-electrode system was fabricated by a simple stencil approach. A carbon-based working electrode was modified using either reduced graphene oxide or molybdenum disulfide nanosheets modified with gold nanoparticle (AuNPs/RGO, AuNPs/MoS2) hybrid structures. The resulting paper-based biosensors offered sensitive detection of miRNA-155 and miRNA-21 by differential pulse voltammetry (DPV) in only 5.0 µL sample. The duration in our assay from the point of electrode modification to the final detection of miRNA was completed within only 35 min. The detection limits for miRNA-21 and miRNA-155 were found to be 12.0 and 25.7 nM for AuNPs/RGO and 51.6 and 59.6 nM for AuNPs/MoS2 sensors in the case of perfectly matched probe-target hybrids. These biosensors were found to be selective enough to distinguish the target miRNA in the presence of single-base mismatch miRNA or noncomplementary miRNA sequences.

The Lateral Flow Immunoassay (LFIA) is by far one of the most successful analytical platforms to perform the on-site detection of target substances. LFIA can be considered as a sort of lab-in-a-hand and, together with other point-of-need tests, has represented a paradigm shift from sample-to-lab to lab-to-sample aiming to improve decision making and turnaround time. The features of LFIAs made them a very attractive tool in clinical diagnostic where they can improve patient care by enabling more prompt diagnosis and treatment decisions. The rapidity, simplicity, relative cost-effectiveness, and the possibility to be used by nonskilled personnel contributed to the wide acceptance of LFIAs. As a consequence, from the detection of molecules, organisms, and (bio)markers for clinical purposes, the LFIA application has been rapidly extended to other fields, including food and feed safety, veterinary medicine, environmental control, and many others. This review aims to provide readers with a 10-years overview of applications, outlining the trends for the main application fields and the relative compounded annual growth rates. Moreover, future perspectives and challenges are discussed.

Hemoglobin A1c (HbA1c) is a standard biomarker to measure long-term average glucose concentration for diagnosis and monitoring of diabetes. Various methods have been reported for measuring HbA1c, however, portable and precise determination is still challenging. Herein, a new highly sensitive electrochemical nanobiosensor is developed for the specific determination of HbA1c. A nanocomposite of reduced graphene oxide (rGO) and gold with hierarchical architecture structure was electrochemically deposited on a cheap and flexible graphite sheet (GS) electrode. The nanocomposite increased the surface area, improved the electron transfer on the electrode surface and augmented the signal. It also provided a suitable substrate for linkage of thiolated DNA aptamer as a bioreceptor on the electrode surface by strong covalent bonding. The quantitative label free detection was carried out by differential pulse voltammetry (DPV) in a phosphate-buffered saline (PBS) solution containing redox probe Fe(CN) 6 3−/4-. The detection is based on insulating the surface in presence of HbA1c and decreasing the current, which is directly related to the HbA1c concentration. The nanobiosensor demonstrated high sensitivity of 269.2 μA. cm−2, wide linear range of 1 nM–13.83 μM with a low detection limit of 1 nM. The biosensor was successfully used for measuring HbA1c in blood real sample. Furthermore, it is promising to use it as a part of a point of care device for low-invasive screening and management of diabetes. Image 1 • An electrochemical nano-genosensor is proposed to hemoglobin A1c determination. • Graphite sheet (GS) is used as a cheap electrode and good candidate to commercialize. • A nanostructure of RGO-gold is electrodeposited on the GS by a facile method. • The biosensor exhibited a very high sensitivity for measuring HbA1c. • The developed biosensor is successfully used for HbA1c measurement in blood sample. [ABSTRACT FROM AUTHOR]

Cerium oxide nanoparticles (NanoCeO 2) and bentonite (Bent) based nanocomposite (CeO 2 -Bent) aqueous sensor drop casted on paper substrate has been used to detect food borne Aflatoxins (AFs) by Curcumin (Cur) functionalized redox reaction mediated colorimetric detection. Optical studies reveal that when chromophore-like Cur is first added to Nano CeO 2 /Bent, it creates a chelate complex that serves as a mediator for enhanced binding with AFs. Cur fictionalized CeO 2 -Bent in the presence of AFB 1 result in a rapid electronic π - π * transition and showed change in color of the dye from red (pH 9.54) to yellow (pH 7). The Hypochromic shift from ∼510 nm to ∼433 nm was plotted linearly at 433 nm for increased AFB 1 level in foods. The calculated LOD indicated values as 0.5 µg/Kg in corn, 2.4 µg/Kg in rice, 0.2 µg/Kg in peanut, 0.7 µg/Kg in almond, 7.8 µg/Kg in arhar and 9.2 µg/Kg in chana respectively. The results were validated with HPLC-FLD obtaining high accuracy and recovery. The sensor reagent further drop casted on paper showed a quick determination for presence of AFs. • A report of CeO 2 -Bent based paper fabricated sensor for food borne aflatoxins. • CeO 2 -Bent showed improved sensitovity in cereals, legumes and oilseeds. • In the presence of AFs hypochromic shift was from 510 nm to 433 nm. • The sensor results are validated by conventional HPLC-FLD technique. • The sensor can be an effective alternative to conventional methods. [ABSTRACT FROM AUTHOR]

In this work, we present a multiplexed (eight simultaneous measurements) paper-based electrochemical device developed in a very simple way and using low-cost materials, such as paper, carbon ink and multifunctional connector headers. Meanwhile, we have also combined the paper-based electrochemical platform with a glass-fiber strip in order to integrate easily a sampling step. Both approaches, simultaneous measuring and sampling, have been applied to the determination of glucose using bienzymatic biosensors. They are fabricated by adsorbing the mixture of enzymes (glucose oxidase and horseradish peroxidase), as well as the ferrocyanide, mediator of the electron transfer, on the paper-based electrode. After drying, the measuring solution (containing either glucose standards or samples) is added and the eight corresponding chronoamperograms are recorded. In the case of the microfluidic approach for sampling purposes, the glass-fiber pad (sampler) is immersed in a container with the solution, which flows by capillarity until it reaches the working electrode. The integration of one more step of the analytical process advances towards real and useful lab-on-a-chip devices. With these designs, a linear range comprised between 0.5 and 15 mM was achieved for glucose determination, with an excellent precision. If the sampler is employed, it is not necessary to use micropipettes and, nevertheless, precise measurements are obtained. The RSD of the slopes obtained for different calibrations performed in different days, with different arrays of electrochemical cells and different solutions is ca. 1%. Accurate results are obtained in the determination of glucose in real samples (orange fruit and cola beverages). Image 1 • Precise sampling is integrated in a paper-based electroanalytical device for glucose biosensing. • Adding a sampler to a paper-based enzymatic sensor advances towards lab-on-paper platforms. • A paper-based simple electrochemical device for eight simultaneous measurements is presented. • A low-cost paper-based sensor samples which analyzes food samples with 8 simultaneous measurements is developed. • Accurate results are obtained for glucose determination in orange fruits and cola beverages. [ABSTRACT FROM AUTHOR]

Abstract Lateral flow biosensors are paper-based devices that allow the detection of different types of analytes with quickness, robustness and selectivity, without leaving behind paper sensors benefits as low-cost, recyclability and sustainability. Nanomaterials have been widely reported in lateral flow biosensors, offering new sensing strategies based on optical or electrical detection techniques. Looking for other advantageous nanomaterials, we propose for the first time the use of iridium oxide (IV) nanoparticles in lateral flow assays for the detection of human immunoglobulin as a model protein. These nanoparticles can be easily prepared and conjugated with biomarkers. Their dark blue color gives a high contrast against the white background of the strips being in this way excellent labels. Graphical abstract fx1 Highlights • Iridium oxide (IV) nanoparticles have been applied for the first time as tags on a lateral flow assay. • Iridium oxide nanoparticles exhibit intense blue color and high biocompatibility with antibodies. • The application of iridium oxide (IV) nanoparticles doubles the sensitivity of the classical gold nanoparticle-based lateral flow. [ABSTRACT FROM AUTHOR]

In the present communication, a unique peroxidase mimicking catalytic property of chitosan was observed and investigated for its analytical application through deployment of a paper based colorimetric sensor for the detection of H 2 O 2 . Chitosan in the presence of H 2 O 2 catalyze the oxidation of TMB to a colored complex proportional to the concentration of H 2 O 2 . Linearity of the chitosan based assay was found to be in the range of 10 μM to 10 mM with a limit of detection and limit of quantification of 1.55 and 1.74 μM, respectively. Analytical performance of chitosan in the form of a paper based sensor demonstrated its potential for point of care applications in determining hydrogen peroxide in a series of analytes. We hypothesize the origin of unusual peroxidase catalytic activity from chitosan with the help of antioxidants and depolymerizing agents. Chitosan breaks H 2 O 2 leading to the formation of highly unstable oxidative species such as hydroxyl ions and free radicals. Catalytic activity of chitosan will overcome the complexity of assays and boost its application in the fabrication of colorimetric diagnostic test kits and in-vivo diagnostics which requires an inexpensive and biocompatible material. [ABSTRACT FROM AUTHOR]

Point-of-care biosensors are important tools developed to aid medical diagnosis and testing, food safety and environmental monitoring. Paper-based biosensors, especially nucleic acid-based lateral flow assays (LFA), are affordable, simple to produce and easy to use in remote settings. However, the sensitivity of such assays to infectious diseases has always been a restrictive challenge. Here, we have successfully electrospun polycaprolactone (PCL) on nitrocellulose (NC) membrane to form a hydrophobic coating to reduce the flow rate and increase the interaction rate between the targets and gold nanoparticles-detecting probes conjugates, resulting in the binding of more complexes to the capture probes. With this approach, the sensitivity of the PCL electrospin-coated test strip has been increased by approximately ten-fold as compared to the unmodified test strip. As a proof of concept, this approach holds great potential for sensitive detection of targets at point-of-care testing. [ABSTRACT FROM AUTHOR]

Paper-based biosensors offer a promising technology to be used at the point of care, enabled by good performance, convenience and low-cost. In this article, we describe a colorimetric vertical-flow DNA microarray (DNA-VFM) that takes advantage of the screening capability of DNA microarrays in a paper format together with isothermal amplification by means of Recombinase Polymerase Amplification (RPA). Different assay parameters such as hybridization buffer, flow rate, printing buffer and capture probe concentration were optimized. A limit of detection (LOD) of 4.4 nM was achieved as determined by tabletop scanning. The DNA-VFM was applied as a proof of concept for detection of Neisseria meningitidis, a primary cause of bacterial meningitis. The LOD was determined to be between 38 and 2.1 × 10 6 copies/VFM assay , depending on the choice of DNA capture probes. The presented approach provides multiplex capabilities of DNA microarrays in a paper-based format for future point-of-care applications. [ABSTRACT FROM AUTHOR]

Electrochemiluminescence (ECL) has been considered to have better performance in rapid detection. A novel paper-based ECL biosensing platform for hepatitis B surface antigen (HBsAg) detection was developed based on a simple paper discs, the flexible design of a machine and a circuit of the custom-made ECL device. Through the modified magnetic suspension sandwich immunoassay protocol, rapid and sensitive clinical HBsAg detection was realized on the developed platform. The platform was characterized using cyclic voltammetry (CV) and ECL methods and obtained a linear range of 34.2 pg/mL–34.2 ng/mL with a 34.2 pg/mL (3σ) detection limit. This paper-based ECL sandwich immunoassay showed high sensitivity and selectivity, acceptable reproducibility, fast analysis time and better performance than chemiluminescent immunoassay (CLIA) and enzyme-linked immunosorbent assay (ELISA) for detecting HBsAg in real clinical serum samples. [ABSTRACT FROM AUTHOR]

Paper-based microfluidic devices are gaining large popularity because of their uncontested advantages of simplicity, cost-effectiveness, limited necessity of laboratory infrastructure and skilled personnel. Moreover, these devices require only small volumes of reagents and samples, provide rapid analysis, and are portable and disposable. Their combination with electrochemical detection offers additional benefits of high sensitivity, selectivity, simplicity of instrumentation, portability, and low cost of the total system. Herein, we present the first example of an integrated paper-based screen-printed electrochemical biosensor device able to quantify nerve agents. The principle of this approach is based on dual electrochemical measurements, in parallel, of butyrylcholinesterase (BChE) enzyme activity towards butyrylthiocholine with and without exposure to contaminated samples. The sensitivity of this device is largely improved using a carbon black/Prussian Blue nanocomposite as a working electrode modifier. The proposed device allows an entirely reagent-free analysis. A strip of a nitrocellulose membrane, that contains the substrate, is integrated with a paper-based test area that holds a screen-printed electrode and BChE. Paraoxon, chosen as nerve agent simulant, is linearly detected down to 3 µg/L. The use of extremely affordable manufacturing techniques provides a rapid, sensitive, reproducible, and inexpensive tool for in situ assessment of nerve agent contamination. This represents a powerful approach for use by non-specialists, that can be easily broadened to other (bio)systems. [ABSTRACT FROM AUTHOR]

In this work we have developed an amperometric enzymatic biosensor in a paper-based platform with a mixed electrode configuration: carbon ink for the working electrode (WE) and metal wires (from a low-cost standard electronic connection) for reference (RE) and auxiliary electrodes (AE). A hydrophobic wax-defined paper area was impregnated with diluted carbon ink. Three gold-plated pins of the standard connection are employed, one for connecting the WE and the other two acting as RE and AE. The standard connection works as a clip in order to support the paper in between. As a proof-of-concept, glucose sensing was evaluated. The enzyme cocktail (glucose oxidase, horseradish peroxidase and potassium ferrocyanide as mediator of the electron transfer) was adsorbed on the surface. After drying, glucose solution was added to the paper, on the opposite side of the carbon ink. It wets RE and AE, and flows by capillarity through the paper contacting the carbon WE surface. The reduction current of ferricyanide, product of the enzymatic reaction, is measured chronoamperometrically and correlates to the concentration of glucose. Different parameters related to the bioassay were optimized, adhering the piece of paper onto a conventional screen-printed carbon electrode (SPCE). In this way, the RE and the AE of the commercial card were employed for optimizing the paper-WE. After evaluating the assay system in the hybrid paper-SPCE cell, the three-electrode system consisting of paper-WE, wire-RE and wire-AE, was employed for glucose determination, achieving a linear range between 0.3 and 15 mM with good analytical features and being able of quantifying glucose in real food samples. [ABSTRACT FROM AUTHOR]

This paper described the development of a small and low cost biosensor consisting of a smartphone-based electrochemical biosensor device and a paper-based biosensor. The device harvested power from the smartphone and transferred data through audio jack. We designed CMOS circuits including a power supply circuit, a potentiostat, and a ΔΣ modulator. The fabrication of a paper-based biosensor was simple: the three electrodes were directly drawn on chromatography paper using a carbon pencil. The paper-based biosensor was low cost, disposable, portable and friendly to the environment. The sensing system was designed to perform the chronoamperometry measurement, and the glucose concentration in a liquid specimen was detected. Results showed that the sensing system was capable of measuring the glucose concentration as precisely as expensive equipments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor. [ABSTRACT FROM AUTHOR]

A new fabrication technology for effective preparation of paper-based biosensors using affinity-binding enzymes is developed. Lactate dehydrogenase (LDH) fused with a cellulose binding domain (CBD) is produced to allow one-step purification and immobilization of the enzyme on paper. Results demonstrate that the binding capacity of the recombinant enzyme is as high as 22 mg-enzyme/g-paper, with much improved stability (by a factor of 25-fold) in comparison to the native parent enzyme. That ensures the solid phase bioassay reactions on surface of the paper sensor are not limited by enzyme availability, and affords much extended sensor shelf life at the same time. The paper sensor shows a linear detection range of 0.5–8 mM of lactate, with changes in color intensity detectable within a much broader concentration range examined (up to 16 mM). The colorimetric display is suited for readouts using either human eyes or hand-held imaging devices such as smart phones. This work promises of a new strategy of developing high performance paper-based sensors for point-of-care diagnosis applications. [ABSTRACT FROM AUTHOR]

Due to the presence of antibiotics in environmental water and their potential influence on the occurrence of antibiotic-resistant bacteria, development of a detection method suitable for the screening of environmental water for antibiotics is required. In this study, we developed a simple colorimetric paper-based biosensor based on a novel principle for the detection of antibiotics inhibiting bacterial protein synthesis, including aminoglycosides, tetracycline, chloramphenicol, and macrolides. This biosensor is based on the detection of a color change induced by β-galactosidase, which is synthesized on freeze-dried paper discs containing an in vitro transcription/translation system. When a water sample without antibiotics is applied to the paper discs, β-galactosidase can be synthesized, and it hydrolyzes a colorimetric substrate, resulting in a color change from yellow to purple. By contrast, in the presence of antibiotics, the color change can be hampered due to an inhibition of β-galactosidase synthesis. We investigated the effect of the incubation temperature and pH of water samples and confirmed that the paper discs showed the color change to purple in the ranges of 15–37°C and pH 6–10. We observed concentration-dependent color variations of the paper discs by the naked eye and further estimated detection limits to be 0.5, 2.1, 0.8, and 6.1 μg/mL for paromomycin, tetracycline, chloramphenicol, and erythromycin, respectively, using digitized pictures. The paper-based biosensor proved to detect 0.5 μg/mL paromomycin, spiked in real environmental water samples, by the naked eye. [ABSTRACT FROM AUTHOR]

Water quality monitoring requires a reliable and practical on-site detection method for heavy metal ions. Combining an in vitro transcription (IVT) technology with allosteric transcription factors (aTFs), we developed a cell-free paper-based biosensor for on-site detection of Hg2+ and Pb2+ in water. Suitable aTFs screened using surface plasmon resonance (SPR) were employed for building biosensors. ATFs could disassociate from DNA due to their specific affinity to metal ions, and fluorescent RNA was transcribed as a signal. The developed biosensor could quantitatively detect Hg2+ in a linear dynamic range of 0.5–500 nM and Pb2+ in a 1–250 nM range in a 1 h period. The LOD of the biosensor was 0.5 nM for Hg2+ and 0.1 nM for Pb2+. The recoveries ranged from 91.09% to 123.24% for actual water samples detection. Furthermore, freeze-drying was used to create a paper-based biosensor that could detect Hg2+ and Pb2+ simultaneously on-site. This research presents a useful technique for various heavy metal ion detections. [Display omitted] • Novel screening strategy of aTFs with BIAcore assay. • The rapid and visual signal output by the 3WJdB aptamer. • High sensitive and selective detection of Hg2+ and Pb2+ by aTFs-based biosensors. • Fast and reliable water quality monitoring and heavy metal risk management. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new paper-based biosensor platform is developed for the analysis of phenylalanine. • The biosensor system is applicable for end-users at point-of-care locations. • The output signal transduction is performed using a regular smartphone. The latest trends in analytical technology are focused on the development of miniaturized, portable systems for on-site analysis of the most important human health biomarkers. These analytical devices should be sensitive, selective, cheap, and simple in usage for the end-point user. Here we describe the construction of a new L-phenylalanine biosensor system based on smartphone-adapted analysis of the fluorescence output. The NAD+-L-phenylalanine dehydrogenase immobilized on silica nanoparticles (200 nm) was used as a biorecognition sensor element and 1-methoxy-5-methylphenazinium methyl sulfate (MPMS) was used as a fluorescent probe. The highly porous fiberglass paper used as a sensor support provides an efficient bioelement load and stabilization while increasing the fluorescence output. The developed biosensor is characterized with accurate L-phenylalanine analysis in the linear frames of 5 – 200 µM and high storage stability (the sensor lost less than 10 % of the initial output during two-month storage). Importantly, the developed technique is very simple and does not depend on the use of any sophisticated analytical instrumentation, since it needs only a UV lamp and a standard smartphone, which makes it very promising for routine analysis of L-phenylalanine. [ABSTRACT FROM AUTHOR]

An electrochemical paper-based sensor was developed for the detection of bacterial infection (BI)-specific biomarker procalcitonin (PCT). Reduced graphene oxide-gold nanoparticles (rGO-AuNP) and poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) were synthesized and were fabricated to a disposable, portable, and inexpensive cellulose fiber paper (CFP) substrate. rGO-AuNP-PEDOT:PSS nanocomposite-modified conductive paper-based biosensing platform was efficaciously fabricated by a constant and simple coating procedure. rGO-AuNP-PEDOT:PSS nanocomposite-modified conductive paper electrode was found to provide a sensitive and conductive substrate for PCT detection. The presence of rGO-AuNP-PEDOT:PSS nanocomposite on CFP substate was investigated by Fourier transform infrared spectrometry, field emission scanning electron microscopy, ultraviolet-visible spectroscopy, and X-ray diffraction studies. The electrochemical behavior of rGO-AuNP-PEDOT:PSS @CFP surface was studied with impedance spectroscopy, cyclic voltammetry, and chronoamperometry techniques. This low-cost paper-based biosensor has a linear range for PCT of 1 × 10 3 to 6 × 10 7  fg mL -1 . This developed sensor exhibited good reproducibility with a relative standard deviation (RSD) of about 3.7%. The proposed CFP-based biosensor has been proven as an accelerated simple point-of-care (POC) exploratory approach for early PCT diagnosis in inadequate areas with limited production facilities, computational techniques, and highly skilled experts., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)