Paper-based Sample Research Articles

A 3D-printed hollow microneedle-based electrochemical sensing device for in situ plant health monitoring

Plant health monitoring is devised as a new concept to elucidate in situ physiological processes. The need for increased food production to nourish the growing global population is inconsistent with the dramatic impact of climate change, which hinders crop health and exacerbates plant stress. In this context, wearable sensors play a crucial role in assessing plant stress. Herein, we present a low-cost 3D-printed hollow microneedle array (HMA) patch as a sampling device coupled with biosensors based on screen-printing technology, leading to affordable analysis of biomarkers in the plant fluid of a leaf. First, a refinement of the 3D-printing method showed a tip diameter of 25.9 ± 3.7 μm with a side hole diameter on the microneedle of 228.2 ± 18.6 μm using an affordable 3D printer (<500 EUR). Notably, the HMA patch withstanded the forces exerted by thumb pressing (i.e. 20-40 N). Subsequently, the holes of the HMA enabled the fluid extraction tested in vitro and in vivo in plant leaves (i.e. 13.5 ± 1.1 μL). A paper-based sampling strategy adapted to the HMA allowed the collection of plant fluid. Finally, integrating the sampling device onto biosensors facilitated the in situ electrochemical analysis of plant health biomarkers (i.e. H2O2, glucose, and pH) and the electrochemical profiling of plants in five plant species. Overall, this electrochemical platform advances precise and versatile sensors for plant health monitoring. The wearable device can potentially improve precision farming practices, addressing the critical need for sustainable and resilient agriculture in changing environmental conditions.

One-step functionalization of paper and simplified antibody immobilization for on-the-spot immunocapture from dried serum in liquid chromatography-tandem mass spectrometry based targeted protein determination.

This work aimed to simplify and improve the process of binding monoclonal antibodies (mAbs) covalently to filter paper for use in dried blood spot sampling, enabling instant capture of protein biomarkers for targeted protein determination. Incorporating the necessary immunocapture sample preparation step in the initial sampling stage saves time and reduces the workload. The biomarker human chorionic gonadotropin (hCG) was used as the model analyte. The antibody-based paper samplers were prepared by functionalizing paper discs (6mm) through a simple reaction using divinyl sulfone (DVS). After DVS activation, the paper discs were incubated with E27 hCG mAbs, followed by 0.05% tween/phosphate buffer saline to block the surface. After sample application and drying, the discs only needed to be washed before tryptic digestion and finally analysed on a nanoliquid chromatography-tandem mass spectrometry system. The finished DVS-mAbs samplers could selectively capture hCG (100 ng/mL) from human serum, with a recovery of 50%. Sample clean-up reduced the number of identified proteins from 132 to 82 before and after wash, respectively, with a 70% reduction in serum albumin signal while still retaining hCG on the sampler during the washing protocol. An evaluation of the samplers revealed excellent linearity (R2 = 0.9995) for hCG in serum with relative standard deviations below 15%. This work has presented the first ever reported paper samplers immobilized with antibodies utilizing DVS chemistry, showing promise in the future of paper-based sampling.

Molecular testing devices for on-site detection of E. coli in water samples

Escherichia coli (E. coli) cells are present in fecal materials that can be the main source for disease‐causing agents in water. As a result, E. coli is recommended as a water quality indicator. We have developed an innovative platform to detect E. coli for monitoring water quality on-site by integrating paper-based sample preparation with nucleic acid isothermal amplification. The platform carries out bacterial lysis and DNA enrichment onto a paper pad through ball-based valves for fluid control, with no need of laboratory equipment, followed by loop-mediated isothermal amplification (LAMP) in a battery-operated coffee mug, and colorimetric detection. We have used the platform to detect E. coli in environmental water samples in about 1 h, with a limit of quantitation of 0.2 CFU/mL, and 3 copies per reaction. The platform was confirmed for detecting multiple E. coli strains, and for water samples of different salt concentrations. We validated the functions of the platform by analyzing recreational water samples collected near the Atlantic Ocean that contain different concentrations of salt and bacteria.

Monitoring disinfection in the Covid-19 era. A reagent-free nanostructured smartphone-based device for the detection of oxidative disinfectants

In this work, a ready-to-use colorimetric device for oxidative disinfectants (i.e., H2O2, C2H4O3, and NaClO) detection has been developed and proved for the evaluation of disinfection treatments in the COVID-19 containment framework.The sensing element of the proposed colorimetric indicator consists of a transparent flexible PET substrate integrating gold and silver nanostructures. The nano-equipped indicator was integrated into different devices realized using a craft-cutting plotter taking advantage of adhesive/thermo-adhesive flexible pouches and filter paper. In particular, two devices are presented (i) a flexible multi-analysis sampling plate and (ii) a paper-based sampling pipette, for the determination of oxidant disinfectants concentration, evaluation of disinfection treatments by spraying/fogging, and for the analysis of disinfectant residues.The sensing strategy relies on the ability of the disinfectants to oxidize the silver nanostructures of the indicator substrate, producing a colorimetric variation monitorable by a smartphone, maximizing the sensitivity using a colored background. The proposed colorimetric assay occurs in 5 min and does not use external reagents; contact of the disinfectant with the sensing surface is the only requirement. The devices had very low limits of detection (∼1 ppb) and good reproducibility (RSD ≤ 2 %, n = 3) for direct disinfectants analysis. They were challenged for the evaluation of indoor disinfection treatments, allowing to evaluate spraying and fogging treatments, as well as residues monitoring.The here proposed system is disposable, low-cost both in therm of material and reagents (0.05 €) and instruments required, portable, and able to return information on the good practices recommended for environmental disinfection.

Paper-based sample processing for the fast and direct MS analysis of multiple analytes from serum samples.

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

A Valve-Enabled Sample Preparation Device with Isothermal Amplification for Multiplexed Virus Detection at the Point-of-Care.

Early and accurate detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the point-of-care is crucial for reducing disease transmission during the current pandemic and future flu seasons. To prepare for potential cocirculation of these two viruses, we report a valve-enabled, paper-based sample preparation device integrated with isothermal amplification for their simultaneous detection. The device incorporates (1) virus lysis and RNA enrichment, enabled by ball-based valves for sequential delivery of reagents with no pipet requirement, (2) reverse transcription loop-mediated isothermal amplification, carried out in a coffee mug, and (3) colorimetric detection. We have used the device for simultaneously detecting inactivated SARS-CoV-2 and influenza A H1N1 viruses in 50 min, with limits of detection at 2 and 6 genome equivalents, respectively. The device was further demonstrated to detect both viruses in environmental samples.

Wearable wristband-based electrochemical sensor for the detection of phenylalanine in biofluids

Wearable electrochemical sensors are driven by the user-friendly capability of on-site detection of key biomarkers for health management. Despite the advances in biomolecule monitoring such as glucose, still, several unmet clinical challenges need to be addressed. For example, patients suffering from phenylketonuria (PKU) should be able to monitor their phenylalanine (PHE) level in a rapid, decentralized, and affordable manner to avoid high levels of PHE in the body which can lead to a profound and irreversible mental disability. Herein, we report a wearable wristband electrochemical sensor for the monitoring of PHE tackling the necessity of controlling PHE levels in PHE hydroxylase deficiency patients. The proposed electrochemical sensor is based on a screen-printed electrode (SPE) modified with a membrane consisting of Nafion, to avoid interferences in biofluids. The membrane also consists of sodium 1,2-naphthoquinone-4-sulphonate for the in situ derivatization of PHE into an electroactive product, allowing its electrochemical oxidation at the surface of the SPE in alkaline conditions. Importantly, the electrochemical sensor is integrated into a wristband configuration to enhance user interaction and engage the patient with PHE self-monitoring. Besides, a paper-based sampling strategy is designed to alkalinize the real sample without the need for sample pretreatment, and thus simplify the analytical process. Finally, the wearable device is tested for the determination of PHE in saliva and blood serum. The proposed wristband-based sensor is expected to impact the PKU self-monitoring, facilitating the daily lives of PKU patients toward optimal therapy and disease management.

Wireless Biosensing System for Daily Self-Testing of Salivary Uric Acid

Here, we report an electrochemical biosensing system capable of performing daily self-testing of uric acid (UA) using saliva. With the aim of daily use, we employed a simplified paper-based sampling device that allows users to collect saliva by themselves, and an amperometric biosensor for the rapid determination of salivary uric acid (SUA) contained in the sampling device without any pretreatment. To utilize the biosensor on site, a wireless control unit with an on-chip potentiostat and Zigbee wireless module was developed. The fundamental characteristics of the biosensor were first evaluated using amperometric techniques. A linear relationship between the concentrations of UA and output currents was confirmed to range from 10 µM to 400 µM. As a real sample test, the biosensor was applied to the measurement of SUA levels in seven healthy male volunteers. The estimated SUA levels were similar to those measured using a commercial UA test kit. Changes in the SUA levels after exercise were also tested. The SUA levels in all the volunteers increased after a bicycle exercise (140 bpm, 10 min). This result was consistent with those obtained in previous studies. Based on the obtained results, our sensor is expected to be useful in the daily self-testing of SUA to improve public health.

Origami paper-based sample preconcentration using sequentially driven ion concentration polarization.

Ion concentration polarization (ICP) is one of the preconcentration techniques which can acquire a high preconcentration factor. Still, the main hurdles of ICP are its instability and low efficiency under physiological conditions with high ionic strength and abundant biomolecules. Here, we suggested a sequentially driven ICP process, which enhanced the electrokinetic force required for preconcentration, enabling enrichment of highly ionic raw samples without increasing the electric field. We acquired a 13-fold preconcentration factor (PF) in human serum using a paper-based origami structure consisting of multiple layers for three-dimensional sequential ICP (3D seq-ICP). Moreover, we demonstrated a paper-based enzyme-linked immunosorbent assay (ELISA) by 3D seq-ICP using tau protein, showing a 6-fold increase in ELISA signals.

Solid reference electrode integrated with paper-based microfluidics for potentiometric ion sensing

A new concept of a solid reference electrode integrated with microfluidic paper-based sampling was introduced and applied in potentiometric ion sensing. The new reference electrode consisted of an Ag/AgCl reference element (reusable) that is in contact with a disposable paper substrate (DPS) containing solid KCl. Thus, no KCl reference solution was applied during the analysis step, but the applied sample solution dissolved the solid KCl in the paper providing the electrolyte for the Ag/AgCl reference element. Such reference electrode was found to give a relatively constant potential after less than ca 1 min of equilibration. Moreover, the response of the reference electrode was not affected by concentrations of background electrolytes in the sample. Thus the proposed reference electrode was integrated with a paper-based microfluidic device and solid-contact ion-selective electrodes (ISEs) sensitive to K+, Na+ and Cl−. The optimized systems were characterized with near-Nernstian sensitivities (59.1 ± 1.5, 57.5 ± 0.5 and –56.4 ± 0.6 mV dec−1) and detection limits (10−4.1 ± 0.1, 10−3.3 ± 0.1 and 10−4.1 ± 0.1 mol dm−3) for K+, Na+ and Cl− ions, respectively. The paper-based measurement setup was favourably used for potentiometric determination of ions in environmental (wastewater sludge) and clinically relevant (sweat) samples.

Sponge and Paper Substrates Coupled with Ion Sensors Utilized in Microfluidic Sampling of High Solid-to-Liquid Ratio Samples

The on-site analysis of any ion concentrations in environmental samples faces many challenges, such as need of sample manipulations and use of a non-portable analytical instrumentation that in turn results in higher uncertainties of the determination and the same time the delay between sampling and the actual measurement. On the other hand, wearable sensors gain more and more attention for instant monitoring of clinically relevant analytes. The portable and relatively low-cost ion-selective electrodes (ISEs) have been extensively studied in context of on-site analysis of ions. However, their potentiometric response majorly depends on the physico-chemical state of an ion-selective membrane (ISM) and the protocol of the analysis. In the situation of nature samples containing high solid-to-liquid ratio, paper substrates and textile were investigated before to integrated with the ISEs to protect the ISM from mechanical damage and the same time deliver micro volumes of liquid to the electrode [1, 2]. Nevertheless, measurements of pH and heavy metals were found to be affected by side reactions and nonspecific adsorption of metal ions to the sampling substrates.In this study, method of pre-modification of paper substrates was investigated to eliminate the detrimental super-Nernstian response otherwise observed [3]. Potentiometric determination of heavy metals in environmental samples (Cd2+ and Pb2+) utilizing paper based microfluidic sampling was studied with a commercial crystalline membrane solid-state Cd2+-and a solid-contact Pb2+-ion selective electrodes. Pre-treatment of paper substrates in the solutions containing the salt with cation the same as the ISEs primary cation, successfully eliminated super Nernstian response. Moreover, the concentration dependent sorption-desorption of metal ion to/from paper substrates was found as possible interferences of using microfluidic paper-based sampling in heavy metal analysis. Then, the amount of heavy metal ions binding onto different substrates was detected.Owning to the lowest heavy metal sorption capacity of PU sponge, microfluidic sponge-based sampling was developed and studied as novel sampling and sample handling substrate to serve as alternative for solution sampling for ion analysis. Possibility of direct measurements of ions in such high solid-to-liquid ratio samples as mud, sludge, wet soil were explored [4].

Conformal, waterproof electronic decals for wireless monitoring of sweat and vaginal pH at the point-of-care

While the monitoring of pH has demonstrated to be an effective technique to monitor an individual's health state, the design of wearable biosensors is subject to critical challenges, such as high fabrication costs, thermal drift, sensitivity to moisture, and the limited applicability for users with metal allergies. This work describes the low-cost fabrication of waterproof electronic decals (WPEDs): highly conformable disposable biosensors capable of monitoring sweat and vaginal pH. WPEDs contain a polyaniline/silver microflakes sensing layer optimized for accurate impedance-based pH quantification across the clinically relevant range of variation of most biofluids. WPEDs also contain a heating layer that serves to both stimulate sweating and prevent saturation of the sensing area, reducing the variability of the measurements. The conformability of WPEDs enables their simple and allergy-free attachment to skin, where they can monitor sweat pH, or to the surface of paper-based sample containers, for the pH-based diagnosis of bacterial vaginosis. WPEDs are mostly transparent, self-adhesive, breathable, flexible, moisture-insensitive, and able to maintain their accuracy under significant mechanical and thermal stresses. A cost-effective wearable and portable impedance analyzer wirelessly transmits pH data in real-time to the smartphone of the user, where a custom-developed App enables long term monitoring and telemedicine applications. Our results demonstrate the feasibility of using inexpensive single-use WPEDs and a reusable, wireless impedance analyzer to provide a wearable solution for the real-time monitoring of sweat pH and the accurate at-home diagnosis of bacterial vaginosis, improving the capabilities of current low-cost, point-of-care diagnostic tests.

Pre-lab proteolysis for dried serum spots – a paper-based sampling concept targeting low abundant biomarkers

Paper-based sampling of biological matrices in combination with mass spectrometry has proven to be a promising technique for bottom-up analysis of proteins.

A disposable paper-based sample clean-up slides for the sensitive determination of trace diethylstilbestrol residues in aquatic products

In this study, a rapid clean-up approach to the microextraction of aquatic food samples based on disposable thin-layer chromatography (dTLC) was developed. First, slides with uniform brushed Gelatin 60 as the stationary phase on paper coated with polyvinyl chloride (PVC) were designed and fabricated successfully. Diethylstilbestrol (DES, mixture of cis- and trans-isomers), which was selected as a typical trace residue of an illegal additive in aquatic products, can be perfectly separated on the paper-based slide by designed solvent development. Under the optimum conditions, the trace amounts of DES in aquatic products can be detected by gas chromatography coupled with mass spectrometer (GC–MS) without obvious interferences from sample matrix. The analytical linearity of DES ranged from 1.0 μg kg−1 to 0.20 mg kg−1, with a detection limit of 0.20 μg kg−1. The present work demonstrated the reliability and applicability of dTLC slides as a preconcentration and clean-up method for the analysis of trace contaminants in food stuff with complex matrix.

All-in-one paper-based sampling chip for targeted protein analysis

A novel all-in-one paper-based sampling concept for mass spectrometric bottom-up protein analysis is here demonstrated in a chip format integrating instant immunocapture, protein reduction, - alkylation and tryptic digestion all in-device. Conventional laboratory grade filter paper was coated with the polymer 2-hydroxyethyl methacrylate-co-2-vinyl-4,4-dimethyl azlactone (pHEMA-VDM) with a subsequent covalent immobilization of the monoclonal antibody E27 targeting the biomarker human chorionic gonadotropin (hCG). In-device protein reduction and alkylation was optimized with regards to reagent concentration and reaction pH. The sampling concept showed a high degree of performance between 10 and 1000 ng/mL (R2 > 0.99) by a five-point calibration curve sampled with hCG spiked to human serum samples and freshly collected whole blood samples, respectively. LOD (experimentally obtained at 100 pg/mL (2.64 pM/0.9 IU/L)) was demonstrated to be up to ten times lower with more than six times faster sample preparation than what has previously been reported for on-paper analysis of hCG in human serum samples.

A Precise Sampling Strip with Microstructures.

This paper presents a precise sampling microstructure formed as a paper strip. We have been developing dialysis system that can be implanted into a human body as an artificial kidney. For the safe use of our artificial kidney, the patients' urine needs to be constantly monitored to detect the abnormal value of ion concentration essential for human life. We are conceiving the monitoring system based on sampling by a paper strip. In this study, we fabricated the strip consisting of slanted and interlocked micropillars for the sampling, known as synthetic microfluidic paper. The paper-like substrate can be fabricated with a well-controlled geometry and subsequently enables precise sampling. Through the conducted experiments, it was shown that synthetic microfluidic paper had better mechanical properties, showed more precision in sampling than paper filter as well as corresponding liquid holding capability to the paper filter. Our proposed paper-based sampling system is expected to lead to the development of minimally invasive ion monitoring system with quantitative sampling strip.

Wax-Assisted One-Step Enzyme-Linked Immunosorbent Assay on Lateral Flow Test Devices.

Lateral flow tests (LFTs) are widely used analytical tools characterized by portability, operator simplicity and short analysis times. A remaining challenge is their limited analytical sensitivity, which in classical immunoassay formats is overcome by enzyme-linked immunosorbent assay (ELISA) formats. The implementation of ELISA to an LFT format however, is hampered by the complexity of the procedure requiring the enzyme substrate addition after sample addition. In this work, a simple method for automation of this procedure without user interference is presented. Originally used sample pads of LFTs have been replaced by hydrophobic wax-modified filter paper-based sample pads to realize a delayed flow a pre-deposited colorimetric ELISA substrate without other alterations to the classical lateral-flow immunoassay format. The performance of the system has been characterized by visualizing flow behavior and final proof-of-concept is provided by a model mouse IgG assay, achieving a limit of detection of 15.8 ng mL-1 from just a single application of the sample solution.

Direct spraying method for fabrication of paper-based microfluidic devices

Direct spraying of hydrophobic materials is an affordable, easy-to-use and equipment-free method for fabrication of flexible microsensors, albeit not yet widely adopted. To explore its application potential, in this paper, we propose and demonstrate two novel hybrid methods to fabricate paper-based components. Firstly, through combing direct spraying with Parafilm embedding, a leak-free paper-based sample preconcentrator for fluorescence sensing was fabricated. The leak-free device worked on the principle of ion concentration polarization (ICP) effect, and achieved enhancement of fluorescent tracer by 220 folds on a paper substrate. Secondly, by using the sprayed hydrophobic patterns, paper-based microsized supercapacitors (mSCs) were fabricated. Vacuum filtration was used to deposit multi-wall carbon nanotubes (MWCNT)-dispersed solution on a porous substrate to form electrodes. A volumetric capacitance of 42.5 mF cm−3 at a current density of 2 mA cm−3 was obtained on the paper-based mSC. Our demonstrations have shown the versatility of direct spraying for the fabrication of integrative paper-based microfluidic devices.

Turning the Page: Advancing Paper-Based Microfluidics for Broad Diagnostic Application.

Infectious diseases are a major global health issue. Diagnosis is a critical first step in effectively managing their spread. Paper-based microfluidic diagnostics first emerged in 2007 as a low-cost alternative to conventional laboratory testing, with the goal of improving accessibility to medical diagnostics in developing countries. In this review, we examine the advances in paper-based microfluidic diagnostics for medical diagnosis in the context of global health from 2007 to 2016. The theory of fluid transport in paper is first presented. The next section examines the strategies that have been employed to control fluid and analyte transport in paper-based assays. Tasks such as mixing, timing, and sequential fluid delivery have been achieved in paper and have enabled analytical capabilities comparable to those of conventional laboratory methods. The following section examines paper-based sample processing and analysis. The most impactful advancement here has been the translation of nucleic acid analysis to a paper-based format. Smartphone-based analysis is another exciting development with potential for wide dissemination. The last core section of the review highlights emerging health applications, such as male fertility testing and wearable diagnostics. We conclude the review with the future outlook, remaining challenges, and emerging opportunities.

Lesbian womens' access to healthcare, experiences with and expectations towards GPs in German primary care.

BackgroundLesbian women have higher rates of physical and psychiatric disorders associated with experiences of discrimination, homophobia and difficulties with coming out. Therefore, easy access to specialized healthcare in an open atmosphere is needed. We aimed to describe women’s access to and experiences with healthcare in Germany, and to assess the responsibility of the general practitioner (GP) compared to other specialities providing primary health care.MethodsA questionnaire study was conducted via internet and paper-based sampling. Using current literature, we designed a questionnaire consisting of sociodemographic data, sexual orientation, access to care and reasons for encounter, disclosure of sexual orientation, experience with the German health system (discrimination, homophobia), and psychological burden. Depression was assessed using the depression screening from the Patient Health Questionnaire (PHQ-2).ResultsWe obtained responses from 766 lesbian women. Although 89% had a primary care physician, only 40% had revealed their sexual orientation to their doctor. The main medical contacts were GPs (66%), gynaecologists (10%) or psychiatrists (6%). Twenty-three percent claimed they were unable to find a primary care physician. Another 12.4% had experienced discrimination. Younger lesbian women with higher education levels and who were less likely to be out to other physicians were more likely to disclose their sexual orientation to their primary care physician. GPs play an important role in healthcare for lesbian women, even in a non-gatekeeping healthcare system like Germany. Study participants suggested improvements regarding gender neutral language, flyers on homosexuality in waiting areas, involvement of partners, training of physicians, directories of homosexual physicians and labelling as a lesbian-friendly practice.ConclusionsGPs should create an open atmosphere and acquire the respective knowledge to provide adequate treatment. Caring for marginal groups should be incorporated in medical training and further education. Ideally, physicians address patients’ sexual orientation pro-actively in order to address individual needs accordingly.Electronic supplementary materialThe online version of this article (doi:10.1186/s12875-016-0562-4) contains supplementary material, which is available to authorized users.