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.

A paper biosensor for overcoming matrix effects interfering with the detection of sputum pyocyanin with competitive immunoassays.

Detecting sputum pyocyanin (PYO) with a competitive immunoassay is a promising approach for diagnosing Pseudomonas aeruginosa respiratory infections. However, it is not possible to perform a negative control to evaluate matrix-effects in competitive immunoassays, and the highly complex sputum matrix often interferes with target detection. Here, we show that these issues are alleviated by performing competitive immunoassays with a paper biosensor. The biosensing platform consists of a paper reservoir, which contains antibody-coated gold nanoparticles, and a substrate containing a competing recognition element, which is a piece of paper modified with an albumin-antigen conjugate. Detection of PYO with a limit of detection of 4.7·10-3 µM and a dynamic range between 4.7·10-1 µM and 47.6 µM is accomplished by adding the sample to the substrate with the competing element and pressing the reservoir against it for 5 min. When tested with patient samples, the biosensor was able to qualitatively differentiate spiked from non-spiked samples, whereas ELISA did not show a clear cut-off between them. Furthermore, the relative standard deviation was lower when determining sputum with the paper-based biosensor. These features, along with a mild liquefaction step that circumvents the use of harsh chemicals or instruments, make our biosensor a good candidate for diagnosing Pseudomonas infections at the bedside through the detection of sputum PYO.

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.

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.

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.

Mobile origami immunosensors for the rapid detection of urinary tract infections.

Urinary tract infections (UTI) have a high prevalence and can yield poor patient outcomes if they progress to urosepsis. Here we introduce mobile origami biosensors that detect UTIs caused by E. coli at the bedside in less than 7 minutes. The origami biosensors are made of a single piece of paper that contains antibody-decorated nanoparticles. When the urine sample contains E. coli, the biosensors generate colored spots on the paper strip. These are then quantified with a mobile app that calculates the pixel intensity in real time. The tests are highly specific and do not cross-react with other common uropathogens. Furthermore, the biosensors only yielded one false negative result when queried with a panel containing 57 urine samples from patients, which demonstrates that they have excellent sensitivity and specificity. This, along with the rapid assay time and smartphone-based detection, makes them useful for aiding in the diagnosis of UTIs at the point of care.

Nanoparticle Reservoirs for Paper-Only Immunosensors.

Biosensors made entirely of paper are becoming increasingly popular due to their low cost, facile fabrication, and lightweight portability for in-field measurements. However, it is difficult to store nanoparticles in paper substrates without irreversibly binding them to the cellulose matrix. This makes it challenging to fabricate biosensors incorporating nanoparticle probes in paper-based reservoirs. Here, we overcome this limitation with a new method for storing protein-decorated nanoparticles on paper substrates that also allows to release them on demand. It consists of spotting nanoparticles onto pieces of filter paper previously modified with polystyrene sulfonate. Gold nanoparticles modified with avidin or antibodies can be easily transferred from the dry reservoir to a receiving wet piece of paper by simply pressing with the finger or a clamp. Paper-based immunosensors incorporating the reservoir enabled the detection of glycoprotein B from human cytomegalovirus in serum with a limit of detection of 0.03 ng mL-1 and a total assay time of only 12 min. The low limit of detection obtained with a short assay time along with the long shelf-life of the reservoirs make the proposed paper-only biosensors ideal of point-of-care diagnostics.