OriPlex: Origami-enabled multiplexed detection of respiratory pathogens.

Origami biosensors leverage paper foldability to develop total analysis systems integrated in a single piece of paper. This capability can also be utilized to incorporate additional features that would be difficult to achieve with rigid substrates. In this article, we report a new design for 3D origami biosensors called OriPlex, which leverages the foldability of filter paper for the multiplexed detection of bacterial pathogens. OriPlex immunosensors detect pathogens by folding nanoparticle reservoirs containing different types of nanoprobes. This releases antibody-coated nanoparticles in a central channel where targets are captured through physical interactions. The OriPlex concept was demonstrated by detecting the respiratory pathogens Pseudomonas aeruginosa (PA) and Klebsiella pneumoniae (KP) with a limit of detection of 3.4·103 cfu mL-1 and 1.4·102 cfu mL-1, respectively, and with a turn-around time of 25 min. Remarkably, the OriPlex biosensors allowed the multiplexed detection of both pathogens spiked into real bronchial aspirate (BAS) samples at a concentration of 105 cfu mL-1 (clinical infection threshold), thus demonstrating their suitability for diagnosing lower tract respiratory infections. The results shown here pave the way for implementing OriPlex biosensors as a screening test for detecting superbugs requiring personalized antibiotics in suspected cases of nosocomial pneumonia.

Facemask analyses for the non-invasive detection of chronic and acute P. aeruginosa lung infections using nanoparticle-based immunoassays.

Pseudomonas aeruginosa (P. aeruginosa) is a pathogen that persistently colonizes the respiratory tract of patients with chronic lung diseases. The risk of acquiring a chronic P. aeruginosa infection can be minimized by rapidly detecting the pathogen in the patient's airways and promptly administrating adequate antibiotics. However, the rapid detection of P. aeruginosa in the lungs involves the analysis of sputum, which is a highly complex matrix that is not always available. Here, we propose an alternative diagnosis based on analyzing breath aerosols. In this approach, nanoparticle immunosensors identify bacteria adhered to the polypropylene layer of a surgical facemask that was previously worn by the patient. A polypropylene processing protocol was optimized to ensure the efficient capture and analysis of the target pathogen. The proposed analytical platform has a theoretical limit of detection of 105 CFU mL-1 in aerosolized mock samples, and a dynamic range between 105 and 108 CFU mL-1. When tested with facemasks worn by patients, the biosensors were able to detect chronic and acute P. aeruginosa lung infections, and to differentiate them from respiratory infections caused by other pathogens. The results shown here pave the way to diagnose Pseudomonas infections at the bedside, as well as to identify the progress from chronic to acute infection.

Plasma-induced nanoparticle aggregation for stratifying COVID-19 patients according to disease severity.

Stratifying patients according to disease severity has been a major hurdle during the COVID-19 pandemic. This usually requires evaluating the levels of several biomarkers, which may be cumbersome when rapid decisions are required. In this manuscript we show that a single nanoparticle aggregation test can be used to distinguish patients that require intensive care from those that have already been discharged from the intensive care unit (ICU). It consists of diluting a platelet-free plasma sample and then adding gold nanoparticles. The nanoparticles aggregate to a larger extent when the samples are obtained from a patient in the ICU. This changes the color of the colloidal suspension, which can be evaluated by measuring the pixel intensity of a photograph. Although the exact factor or combination of factors behind the different aggregation behavior is unknown, control experiments demonstrate that the presence of proteins in the samples is crucial for the test to work. Principal component analysis demonstrates that the test result is highly correlated to biomarkers of prognosis and inflammation that are commonly used to evaluate the severity of COVID-19 patients. The results shown here pave the way to develop nanoparticle aggregation assays that classify COVID-19 patients according to disease severity, which could be useful to de-escalate care safely and make a better use of hospital resources.

Improving the Quantification of Colorimetric Signals in Paper-Based Immunosensors with an Open-Source Reader.

Measuring the colorimetric signals produced by the biospecific accumulation of colorimetric probes and recording the results is a key feature for next-generation paper-based rapid tests. Manual processing of these tests is time-consuming and prone to a loss of accuracy when interpreting faint and patchy signals. Proprietary, closed-source readers and software companies offering automated smartphone-based assay readings have both been criticized for interoperability issues. Here, we introduce a minimal reader prototype composed of open-source hardware and open-source software that has the benefits of automatic assay quantification while avoiding the interoperability issues associated with closed-source readers. An image-processing algorithm was developed to automate the selection of an optimal region of interest and measure the average pixel intensity. When used to quantify signals produced by lateral flow immunoassays for detecting antibodies against SARS-CoV-2, results obtained with the proposed algorithm were comparable to those obtained with a manual method but with the advantage of improving the precision and accuracy when quantifying small spots or faint and patchy signals.

Dissolvable Polymer Valves for Sweat Chrono-Sampling in Wearable Paper-Based Analytical Devices.

Paper sensors with colorimetric signal transduction mechanisms are promising for developing single-use wearable patches that only require a smartphone to quantify signals. However, measuring biomarker fluctuations with colorimetric wearable sensors requires implementing a chrono-sampling method for performing sequential measurements. In this article, we report on a chrono-sampling method that enables the fabrication of wearable devices made entirely of filter paper. It consists of using dried polymers as closed valves that deflect the flow of liquids to different transducers of a multisensor. As time passes by, the polymer dissolves and the valve opens. The sequential opening of the valves results in a succession of measurements that reveals fluctuations in the concentration of the target analyte. This concept was demonstrated with a paper multisensor capable of performing nine consecutive pH measurements. The device was also adapted for developing a urea biosensor that detects pH measurements generated by the hydrolysis of the analyte catalyzed by urease. The proposed analytical platform could monitor the pH of sweat with an accuracy and precision comparable to a laboratory-based method when worn during an exercise routine. The results shown here pave the way for developing colorimetric wearable biosensors that measure variations in the concentration of biomarkers such as glucose, lactate, creatinine, or uric acid over time.

Micro- and nanosensors for detecting blood pathogens and biomarkers at different points of sepsis care.

Severe infections can cause a dysregulated response leading to organ dysfunction known as sepsis. Sepsis can be lethal if not identified and treated right away. This requires measuring biomarkers and pathogens rapidly at the different points where sepsis care is provided. Current commercial approaches for sepsis diagnosis are not fast, sensitive, and/or specific enough for meeting this medical challenge. In this article, we review recent advances in the development of diagnostic tools for sepsis management based on micro- and nanostructured materials. We start with a brief introduction to the most popular biomarkers for sepsis diagnosis (lactate, procalcitonin, cytokines, C-reactive protein, and other emerging protein and non-protein biomarkers including miRNAs and cell-based assays) and methods for detecting bacteremia. We then highlight the role of nano- and microstructured materials in developing biosensors for detecting them taking into consideration the particular needs of every point of sepsis care (e.g., ultrafast detection of multiple protein biomarkers for diagnosing in triage, emergency room, ward, and intensive care unit; quantitative detection to de-escalate treatment; ultrasensitive and culture-independent detection of blood pathogens for personalized antimicrobial therapies; robust, portable, and web-connected biomarker tests outside the hospital). We conclude with an overview of the most utilized nano- and microstructured materials used thus far for solving issues related to sepsis diagnosis and point to new challenges for future development.

Nanoparticle transfer biosensors for the non-invasive detection of SARS-CoV-2 antigens trapped in surgical face masks.

Detecting SARS-CoV-2 antigens in respiratory tract samples has become a widespread method for screening new SARS-CoV-2 infections. This requires a nasopharyngeal swab performed by a trained healthcare worker, which puts strain on saturated healthcare services. In this manuscript we describe a new approach for non-invasive COVID-19 diagnosis. It consists of using mobile biosensors for detecting viral antigens trapped in surgical face masks worn by patients. The biosensors are made of filter paper containing a nanoparticle reservoir. The nanoparticles transfer from the biosensor to the mask on contact, where they generate colorimetric signals that are quantified with a smartphone app. Sample collection requires wearing a surgical mask for 30 min, and the total assay time is shorter than 10 min. When tested in a cohort of 27 patients with mild or no symptoms, an area under the receiving operating curve (AUROC) of 0.99 was obtained (96.2 % sensitivity and 100 % specificity). Serial measurements revealed a high sensitivity and specificity when masks were worn up to 6 days after diagnosis. Surgical face masks are inexpensive and widely available, which makes this approach easy to implement anywhere. The excellent sensitivity, even when tested with asymptomatic patient samples, along with the mobile detection scheme and non-invasive sampling procedure, makes this biosensor design ideal for mass screening.

Detection of low glucose levels in sweat with colorimetric wearable biosensors.

Low glucose levels during exercise may lead to hypoglycemia, which can have grave consequences in diabetic athletes. Mobile colorimetric wearable biosensors that measure glucose levels in sweat are ideal for self-monitoring as they can utilize the camera in smartphones for signal reading. However, colorimetric biosensors proposed thus far have higher limit of detection (LOD) than electrochemical devices, which makes them unsuitable for detecting hypoglycemia. In this manuscript we describe colorimetric wearable biosensors that detect glucose in sweat with an LOD of 0.01 mM and a dynamic range up to 0.15 mM. The devices are made of filter paper and incorporate a sweat volume sensor and a color chart for signal correction. The biosensors do not suffer from interferences originated by delayed sample readings, or differences in bending angle and sample pH. When applied to volunteers performing an exercise routine, sweat glucose levels corrected with sweat volume measurements correlated well with blood glucose measurements performed with a commercial device. The devices are lightweight and easily disposable. These features, along with the smartphone-based colorimetric readout, makes them promising as "over-the-counter" tests for measuring glucose levels non-invasively during exercise.

Paper biosensors for detecting elevated IL-6 levels in blood and respiratory samples from COVID-19 patients.

Decentralizing COVID-19 care reduces contagions and affords a better use of hospital resources. We introduce biosensors aimed at detecting severe cases of COVID-19 in decentralized healthcare settings. They consist of a paper immunosensor interfaced with a smartphone. The immunosensors have been designed to generate intense colorimetric signals when the sample contains ultralow concentrations of IL-6, which has been proposed as a prognosis biomarker of COVID-19. This is achieved by combining a paper-based signal amplification mechanism with polymer-filled reservoirs for dispensing antibody-decorated nanoparticles and a bespoken app for color quantification. With this design we achieved a low limit of detection (LOD) of 10-3 pg mL-1 and semi-quantitative measurements in a wide dynamic range between 10-3 and 102 pg mL-1 in PBS. The assay time is under 10 min. The low LOD allowed us to dilute blood samples and detect IL-6 with an LOD of 1.3 pg mL-1 and a dynamic range up to 102 pg mL-1. Following this protocol, we were able to stratify COVID-19 patients according to different blood levels of IL-6. We also report on the detection of IL-6 in respiratory samples (bronchial aspirate, BAS) from COVID-19 patients. The test could be easily adapted to detect other cytokines such as TNF-α and IL-8 by changing the antibodies decorating the nanoparticles accordingly. The ability of detecting cytokines in blood and respiratory samples paves the way for monitoring local inflammation in the lungs as well as systemic inflammation levels in the body.

Wearable Analytical Platform with Enzyme-Modulated Dynamic Range for the Simultaneous Colorimetric Detection of Sweat Volume and Sweat Biomarkers.

In this manuscript, we introduce a wearable analytical platform that simultaneously measures the concentration of sweat lactate and sample volume. It contains two sensors entirely made of filter paper that can be easily affixed on the skin with medical-grade tape. The lactate biosensor features a unique signal modulation mechanism that enables fine-tuning the dynamic range. It consists of adding a competitive enzyme inhibitor in different reservoirs. Thanks to this, it is possible to choose between a very low limit of detection (0.06 mM) and a linear response in the physiological concentration range (10-30 mM). The sweat volume sensor was obtained by adding a reservoir containing gold nanoparticles. As the wearer sweats, the nanoparticles are carried through a paper channel. This is used to gauge the volume of sample by measuring the distance traveled by the nanoprobes. Using fine-tuned lactate biosensors and combining them with the volume sensors allowed us to quantify variations in the levels of sweat lactate independently of the wearer's sweat rate during an exercise routine. The platform design can be customized to meet the end user's needs, which makes it ideal for developing a wide array of disposable wearable biosensors.