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Infections caused by bacteria that produce β-lactamases (BLs) are a major problem in hospital settings. The phenotypic detection of these bacterial strains requires culturing samples prior to analysis. This procedure may take up to 72 h, and therefore it cannot be used to guide the administration of the first antibiotic regimen. Here, we propose a multisensor for identifying pathogens bearing different types of β-lactamases above the infectious dose threshold within 90 min that does not require culturing samples. Instead, bacterial cells are preconcentrated in the cellulose scaffold of a paper-based multisensor. Then, 12 assays are performed in parallel to identify whether the pathogens produce carbapenemases and/or cephalosporinases, including metallo-β-lactamases, extended-spectrum β-lactamases (ESBLs), and AmpC enzymes. The multisensor generates an array of colored spots that can be quantified with image processing software and whose interpretation leads to the detection of the different enzymes depending on their specificity toward the hydrolysis of certain antibiotics, and/or their pattern of inhibition or cofactor activation. The test was validated for the diagnosis of urinary tract infections. The inexpensive paper platform along with the uncomplicated colorimetric readout makes the proposed prototypes promising for developing fully automated platforms for streamlined clinical diagnosis.

Lung IL-6 is a promising biomarker for predicting respiratory failure during pulmonary infections. This biomarker is found in respiratory samples which need to be liquefied prior to analysis. Traditional liquefying methods use reducing agents such as dithiothreitol (DTT). However, DTT impairs immunodetection and does not liquefy highly viscous samples. We propose an enzymatic method that liquefies samples by means of generating O2 bubbles with endogenous catalase. Low respiratory tract specimens from 48 mechanically ventilated patients (38 with SARS-CoV-2 infection) were treated with DTT or with the enzymatic method. We used turbidimetry to compare the liquefaction degree and IL-6 was quantified with ELISA. Finally, we used AUC-ROC, time-to-event and principal component analysis to evaluate the association between respiratory compromise or local inflammation and IL-6 determined with both methods. Enzymatically treated samples were better liquefied than those reduced by DTT, which resulted in higher ELISA signals. Lung IL-6 levels obtained with the enzymatic procedure were negatively correlated with the oxygenation index (PaO2/FiO2) and the time of mechanical ventilation. The proposed enzymatic liquefaction method improves the sensitivity for lung IL-6 detection in respiratory samples, which increases its predictive power as a biomarker for evaluating respiratory compliance.

Respiratory infections caused by multi-drug-resistant Pseudomonas aeruginosa often yield poor outcomes if not detected right away. However, detecting this pathogen in respiratory samples with a rapid diagnostic test is challenging because the protective biofilms created by the pathogen are themselves surrounded by a high-viscosity sputum matrix. Here, we introduce a method for liquefying respiratory samples and disrupting bacterial biofilms on the spot within a minute. It relies on the generation of oxygen bubbles by bacterial catalase through the addition of hydrogen peroxide. When coupled with a mobile biosensor made of paper, the resulting diagnostic kit was able to detect P. aeruginosa infections in sputa from patients with excellent sensitivity and specificity within 8 min. The quick turnaround time along with few infrastructure requirements make this method ideal for the rapid screening of P. aeruginosa infections at the point of 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. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00604-022-05171-2.

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.

Hyperdegranulation of neutrophilic granulocytes is a common finding in sepsis that directly contributes to the heightened immune response leading to organ dysfunction. Currently, cell degranulation is detected by flow cytometry, which requires large infrastructure that is not always available at the point of care. Here, we propose a plasmonic assay for detecting the degranulation status of septic cells colorimetrically. It is based on triggering the aggregation of gold nanoparticles with cationic granule proteins. Cells from septic patients contain fewer granules and therefore release less cationic proteins than healthy cells. This results in red-colored assays than can be easily detected by eye. The assay can selectively detect cationic granule proteins even in the presence of an excess of unrelated proteins, which is key to detect degranulation with high specificity. Coupling this signal generation mechanism with a magnetic purification step enabled the identification of septic cells with the same performance as flow cytometry. This makes the proposed method a promising alternative for diagnosing sepsis in decentralized healthcare schemes.

Lung IL-6 is a promising biomarker for predicting respiratory failure during pulmonary infections. This biomarker is found in respiratory samples which need to be liquefied prior to analysis. Traditional liquefying methods use reducing agents such as dithiothreitol (DTT). However, DTT impairs immunodetection and does not liquefy highly viscous samples. We propose an enzymatic method that liquefies samples by means of generating O

Herein, we present an optimized bottom-up approach for the fabrication of homogeneous small spherical silver nanoparticles, with average diameter sizes ranging from 10 to 30 nm and the associated p...

We report on the bioinspired growth of gold nanoparticles (GNPs) in biocompatible hydrogels to develop plasmonic hybrid materials. The new hydrogel (CS-Sq) is prepared from chitosan and diethylsquarate and is formed via noncovalent interactions rising between the in situ formed ionic squaric acid derivatives and chitosan. Interestingly, when the hydrogel is prepared in the presence of HAuCl4, GNPs with controlled sizes between 15 and 50 nm are obtained, which are homogeneously distributed within the plasmonic hydrogels (GNPs-CS-Sq). We found that the supramolecular nature and the composition of the CS-Sq hydrogels are key for the growth process of GNPs where the squaric derivatives act as reducing agents and the chitosan hydrogel network provides nucleation points and supports the GNPs. Accordingly, the hydrogel acts as a bioinspired reactor and permits to gain certain control on the size of GNPs by adjusting the concentration of chitosan and HAuCl4. Besides the intrinsic and tunable plasmonic properties of the GNPs-CS-Sq hydrogels, it was found that the gels could be useful as heterogeneous catalysts for organic reactions. Furthermore, cell viability studies indicate that the new hydrogels exhibit suitable biocompatibility. Thus, the proposed method for obtaining GNPs-CS-Sq hydrogels has the potential for the development of a wide variety of other hybrid chitosan materials useful for catalysis, biosensing, cell culture, tissue engineering, and drug delivery applications.

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.

Identifying the pathogen responsible for an infection is a requirement in order to personalize antimicrobial treatments. Detecting bacterial enzymes, such as proteases, lipases, and oxidoreductases, is a winning approach for detecting pathogens at the point of care. In this Article, a new method for detecting urease-producing bacteria rapidly and at ultralow concentrations is reported. In this method, longsome bacteriological culture steps are substituted for a 10 min capture procedure with positively charged magnetic beads. The presence of urease-positive bacteria on the particles is then queried with a plasmonic signal generation step that generates blue- or red-colored nanoparticle suspensions upon addition of the enzyme substrate. These colorimetric signals, which can be easily identified by eye, are generated by the NH3-dependent assembly of gold nanoparticles in the presence of bovine serum albumin (BSA). The proposed method can detect Proteus mirabilis with a limit of detection of 101 cells mL-1, with a total assay time of 40 min, even in the presence of a large excess of urease-negative bacteria ( Pseudomonas aeruginosa). Furthermore, it does not require bulky equipment, and it can detect P. mirabilis at clinically relevant concentrations within minutes, making it suitable for detecting urease-positive pathogens at the point of care.

Graphical abstract, 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 minutes, 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.

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.

Lung-secreted IgG and IgM antibodies are valuable biomarkers for monitoring the local immune response against respiratory infections. These biomarkers are found in lower airway secretions that need to be liquefied prior to analysis. Traditional methods for sample liquefaction rely on reducing disulfide bonds, which may damage the structure of the biomarkers and hamper their immunodetection. Here, we propose an alternative enzymatic method that uses O2 bubbles generated by endogenous catalase enzymes in order to liquefy respiratory samples. The proposed method is more efficient for liquefying medium- and high-viscosity samples and does not fragment the antibodies. This prevents damage to antigen recognition domains and recognition sites for secondary antibodies that can decrease the signal of immunodetection techniques. The suitability of the enzymatic method for detecting antibodies in respiratory samples is demonstrated by detecting anti-SARS-CoV-2 IgG and IgM to viral N-protein with gold standard ELISA in bronchial aspirate specimens from a multicenter cohort of 44 COVID-19 patients. The enzymatic detection sharply increases the sensitivity toward IgG and IgM detection compared to the traditional approach based on liquefying samples with dithiothreitol. This improved performance could reveal new mechanisms of the early local immune response against respiratory infections that may have gone unnoticed with current sample treatment methods.

Graphical abstract, Highlights • Detection of cytokine storm biomarkers with mobile biosensors. • Immunosensors are made solely of cellulose modified with antibody-decorated nanoparticles. • Turnaround time under 10 min. • Detection of biomarkers in respiratory samples allows monitoring local inflammation. • Compatible with decentralized health care schemes., 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.

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.

Respiratory infections caused by multi-drug-resistant

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., Dr de la Rica acknowledges financial support from grants FEDER/Ministerio de Ciencia, Innovacion y Universidades/Agencia Estatal de Investigacion/-Proyecto CTQ2017-82432-R and FEDER/Ministerio de Ciencia, Innovacion y Universidades/Agencia Estatal de Investigacion/-Proyecto CTQ2017-92226-EXP as well as a Radix fellowship from IdISBa/Impost turisme sostenible/Govern de les Illes Balears.

The global COVID-19 pandemic has oversaturated many intensive care units to the point of collapse, leading to enormous spikes in death counts. Before critical care becomes a necessity, identifying patients who are likely to become critically ill and providing prompt treatment is a strategy to avoid ICU oversaturation. There is a consensus that a hyperinflammatory syndrome or a "cytokine storm" is responsible for poor outcomes in COVID-19. Measuring cytokine levels at the point of care is required in order to better understand this process. In this Perspective, we summarize the main events behind the cytokine storm in COVID-19 as well as current experimental treatments. We advocate for a new biosensor-enabled paradigm to personalize the management of COVID-19 and stratify patients. Biosensor-guided dosing and timing of immunomodulatory therapies could maximize the benefits of these anti-inflammatory treatments while minimizing deleterious effects. Biosensors will also be essential in order to detect complications such as coinfections and sepsis, which are common in immunosuppressed patients. Finally, we propose the ideal features of these biosensors using some prototypes from the recent literature as examples. Multisensors, lateral flow tests, mobile biosensors, and wearable biosensors are seen as key players for precision medicine in COVID-19.

There is limited information available describing the clinical and epidemiological features of Spanish patients requiring hospitalization for coronavirus disease 2019 (COVID-19). In this observational study, we aimed to describe the clinical characteristics and epidemiological features of severe (non-ICU) and critically patients (ICU) with COVID-19 at triage, prior to hospitalization. Forty-eight patients (27 non-ICU and 21 ICU) with positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were analyzed (mean age, 66 years, [range, 33&ndash, 88 years], 67% males). There were no differences in age or sex among groups. Initial symptoms included fever (100%), coughing (85%), dyspnea (76%), diarrhea (42%) and asthenia (21%). ICU patients had a higher prevalence of dyspnea compared to non-ICU patients (95% vs. 61%, p = 0.022). ICU-patients had lymphopenia as well as hypoalbuminemia. Lactate dehydrogenase (LDH), C-reactive protein (CRP), and procalcitonin were significantly higher in ICU patients compared to non-ICU (p &lt, 0.001). Lower albumin levels were associated with poor prognosis measured as longer hospital length (r = &minus, 0.472, p &lt, 0.001) and mortality (r = &minus, 0.424, p = 0.003). As of 28 April 2020, 10 patients (8 ICU and 2 non-ICU) have died (21% mortality), and while 100% of the non-ICU patients have been discharged, 33% of the ICU patients still remained hospitalized (5 in ICU and 2 had been transferred to ward). Critically ill patients with COVID-19 present lymphopenia, hypoalbuminemia and high levels of inflammation.

The dysregulated release of cytokines has been identified as one of the key factors behind poorer outcomes in COVID-19. This cytokine storm produces an excessive inflammatory and immune response, especially in the lungs, leading to acute respiratory distress (ARDS), pulmonary edema and multi-organ failure. Alleviating this inflammatory state is crucial to improve prognosis. Pro-inflammatory factors play a central role in COVID-19 severity, especially in patients with comorbidities. In these situations, an overactive, untreated immune response can be deadly, suggesting that mortality in COVID-19 cases is likely due to this virally driven hyperinflammation. Administering immunomodulators has not yielded conclusive improvements in other pathologies characterized by dysregulated inflammation such as sepsis, SARS-CoV-1, and MERS. The success of these drugs at reducing COVID-19-driven inflammation is still anecdotal and comes with serious risks. It is also imperative to screen the elderly for risk factors that predispose them to severe COVID-19. Immunosenescence and comorbidities should be taken into consideration. In this review, we summarize the latest data available about the role of the cytokine storm in COVID-19 disease severity as well as potential therapeutic approaches to ameliorate it. We also examine the role of inflammation in other diseases and conditions often comorbid with COVID-19, such as aging, sepsis, and pulmonary disorders. Finally, we identify gaps in our knowledge and suggest priorities for future research aimed at stratifying patients according to risk as well as personalizing therapies in the context of COVID19-driven hyperinflammation., RR acknowledges funding from IdISBa/Impost turisme sostenible/Govern de les Illes Balears (Agencia d'Estrategia Turistica de les Illes Balears-Govern de les Illes Balears) through a Radix fellowship. MG-F acknowledges funding by the Miguel Servet Program (MS19/00201), Instituto de Salud Carlos III (ISCIII), Madrid. Figures were created with images from Canva (www.canva.com) under Free Media License Agreement.

Detecting small variations in the levels of IL-6 is crucial for the early diagnosis of sepsis. To be useful in clinical decision-making, this requires detecting IL-6 rapidly in whole blood and with portable readers. Here we introduce immunosensors made of filter paper that use plasmonic nanoprobes to detect IL-6 rapidly in unprocessed blood with an unmodified smartphone. Key aspects of the biosensor fabrication were optimized in order to reduce the assay time without losing sensitivity. This included testing three bioconjugation routes for protein attachment to nanoprobes using gold nanoparticles covered with carboxylate or amine moieties, or polyvinylpyrrolidone (PVP), as starting materials, and using alternating layers of polyelectrolytes to bind the capture antibody to the paper substrate. Smartphone-based signal quantification was achieved with a custom-made app featuring a unique augmented reality guidance system that circumvents the need for smartphone attachments and automates all the steps involved in color quantification. The biosensors were able to detect IL-6 with a limit of detection of 0.1 pg mL(-1) and a total assay time within 17 min. They could also detect an increase in IL-6 of only 12.5 pg mL(-1) over basal levels in whole blood with 99% confidence. The high sensitivity and rapid turnaround time afforded by the optimized biosensors and the fully automated real-time densitometry app make our biosensors well suited for emergency healthcare situations such as the identification of potential sepsis cases., R. R. acknowledges financial support from FEDER/Ministerio de Ciencia, Innovacion y Universidades/Agencia Estatal de Investigacion/- Proyecto CTQ2017-82432-R (biosensor development) and Proyecto CTQ2017-92226-EXP (app development). R. R. and S. M. R. acknowledge funding from a Radix fellowship from IdISBa/Impost turisme sostenible/Govern de les Illes Balears. A. A. acknowledges a fellowship for doctoral studies from Roche (Stop fuga de cerebros program). R. A. A.-P. and N. P. P. acknowledge financial support from Spanish Ministerio de Economia y Competitividad (CTQ2017-88648R and RYC-2015-19107), the Generalitat de Cataluna (2017SGR883), the Universitat Rovira i Virgili (2018PFR-URV-B2-02), and the Universitat Rovira i Virgili and Banco Santander (2017EXIT-08).

Signal amplification in colorimetric biosensors usually requires labile reagents and/or additional steps, which are not ideal for in-field measurements. Here we show that the limit of detection of a gold nanoparticle-labeled colorimetric immunosensor can be decreased 10-fold by performing the assay on a folded piece of paper. In this approach, the intrinsic porosity of the substrate enables generating multiple signals with a single drop of reagent. This yields combined signals with higher optical density thanks to the semitransparent nature of the wet material. It will be shown that this method can decrease the limit of detection 10 times in a model immunosensor for the detection of immunoglobulins. The procedure is so straightforward that it could be easily implemented to decrease the limit of detection of microfluidic paper-based analytical devices with colorimetric readouts.

Detecting variations in the localized surface plasmon resonance (LSPR) of gold nanoparticles is a widespread approach for developing colorimetric biosensors. This is usually performed with a large spectrophotometer, which is not suitable for in-field measurements, or with the naked eye, which only allows detecting large spectral variations leading to clear changes in color. Here we demonstrate that patterns printed on paper can transduce LSPR variations caused by the aggregation of plasmonic nanosensors using a mobile device as the reader. The sensitivity of the proposed paper transducers is tested with a new enzyme-less signal generation mechanism for biosensors based on triggering the aggregation of gold nanoparticles in the presence of neutravidin. A competitive immunoassay using this mechanism and the proposed transducers can detect the model analyte C-reactive protein with a limit of detection of 3·10−8 g mL−1 within 1 h, which is comparable to an ELISA using a spectrophotometer to detect the signal. The fabrication of the transducers only requires a common toner printer, and the signal can be detected with the ubiquitous smartphone using a downloadable app, which makes the proposed detection platform ideal for developing mobile biosensors for global health applications.

Temperature is a crucial parameter in biology, nanoelectronics, nanophotonics, and microfluidics. Optical methods excel for measuring temperature because they are noninvasive, spatially accurate, and can measure real time local changes in temperature. Among these, fluorescence anisotropy-based methods are particularly advantageous because they are less affected by changes in the probe concentration and irradiation conditions. However, at physiologically relevant temperature ranges in aqueous solution, fluorescence anisotropy contrast can only be achieved with rather large fluorescent proteins such as the green fluorescent protein (GFP), which can limit the range of applications through this method. Here, we propose a method to add thermosensitivity to any protein thereby transforming them into fluorescence anisotropy-based thermoprobes. It consists of covalently attaching a dye to the protein, which increases the rotational time of the dye-protein system compared to the free dye and confers thermosensitiv...

Gold nanoparticles (GNPs) have become essential tools used in nanobiotechnology due to their tunable plasmonic properties and low toxicity in biological samples. Among the available approaches for imaging GNPs internalized by cells, hyperspectral techniques stand out due to their ability to simultaneously image and perform spectral analysis of GNPs. Here, we present a study utilizing a recently introduced hyperspectral imaging technique, live-cell PWS, for the imaging, tracking, and spectral analysis of GNPs in live cancer cells. Using principal components analysis, the extracellular or intracellular localization of the GNPs can be determined without the use of exogenous labels. This technique uses wide-field white light, assuring minimal toxicity and suitable signal-to-noise ratio for spectral and temporal resolution of backscattered signal from GNPs and local cellular structures. The application of live-cell PWS introduced here could make a great impact in nanomedicine and nanotechnology by giving new insights into GNP internalization and intracellular trafficking.

Colorimetric tests are becoming increasingly popular in point-of-need analyses due to the possibility of detecting the signal with the naked eye, which eliminates the utilization of bulky and costly instruments only available in laboratories. However, colorimetric tests may be interpreted incorrectly by nonspecialists due to disparities in color perception or a lack of training. Here we solve this issue with a method that not only detects colorimetric signals but also interprets them so that the test outcome is understandable for anyone. It consists of an augmented reality (AR) app that uses a camera to detect the colored signals generated by a nanoparticle-based immunoassay, and that yields a warning symbol or message when the concentration of analyte is higher than a certain threshold. The proposed method detected the model analyte mouse IgG with a limit of detection of 0.3 μg mL–1, which was comparable to the limit of detection afforded by classical densitometry performed with a nonportable device. Whe...

In this paper, we report on a new type of self-assembled plasmonic nanostructure, called gold suprashells, which are assembled around superparamagnetic iron oxide nanoparticle (SPION) cores. Gold suprashells have multiple surface plasmon resonances over a broad vis–NIR wavelength range, which makes them useful in applications where broadband absorption is required. For example, suprashells are efficient substrates that enhance surface-enhanced Raman scattering signals across multiple excitation wavelengths. This unique multiresonant character is afforded by the suprashell structure, which comprises anisotropic assemblies of nanoparticles of tunable length. Furthermore, gold suprashells generate more heat when excited with a laser compared to the nanoparticle building blocks, therefore making them promising materials for photothermal applications. The suprashells can potentially be assembled onto any negatively charged core, which opens up multiple possibilities for the development of multifunctional core/...

Self-propelled particles are revolutionizing sensing applications thanks to a unique motion-based signal generation mechanism in which biorecognition reactions are detected as changes in the velocity of the colloids. Here a new family of self-propelled multifunctional Janus particles is introduced that enables detecting changes in particle motion colorimetrically. The particles consist of an iron oxide core that provides color and magnetism, and a Janus coating that provides biospecific recognition and locomotive properties. In this approach, biomolecular interactions trigger changes in particle motion that are detected as variations in color when spotted on a piece of paper. These variations in color are then read and quantified with a custom-made smartphone app. The high surface area and magnetism of the particles makes them ideal building blocks for developing biosensors because they allow for the rapid capture of a target molecule and the removal of non-specific interactions. Biosensors engineered with the proposed multifunctional particles were able to detect the sepsis biomarker procalcitonin at clinically relevant concentrations within 13 min in whole blood, which is faster than other approaches requiring hour-long incubation steps under controlled conditions to detect the same biomarker in purified serum. The short assay time along with the point-of-need design makes these biosensors suitable for stratifying patients according to their sepsis risk level during triage independently of resource constraints.

Providing the right antibiotic treatment as soon as possible is crucial to improve sepsis outcomes. In this context, infections by bacteria resistant to β-lactams can be deadly. Current approaches for identifying these pathogens are longsome and require complex infrastructure. Here we introduce a diagnostic kit for detecting carbapenemase-producing pathogens at the bedside. It generates colored tests that are easily distinguished by eye or by analyzing a smartphone photograph. The color is generated by gold nanoparticles, which have been designed to detect β-lactamase activity robustly and with ultra-high sensitivity. Neither microbial culture nor specialized equipment are required. The kit can detect infections by carbapenemase-producing bacteria directly from urine samples within 2.5 h, even when these contain an excess of interfering bacteria. It can also detect them in sputum samples within 3 h. The rapid assay time coupled with the ability to detect hazardous infections enables the personalization of antimicrobial treatments much faster than a full antibiotic susceptibility test, which often takes several days to be accomplished. This kit can also be implemented in resource-constrained scenarios where other options for antimicrobial susceptibility testing may not be available.

Colorimetric detection with smartphones is ideal for point-of-care measurements because the signal reader is easily available. Densitometric detection schemes enable semiquantitative measurements but require a lightproof box to control photographic conditions and/or extensive data treatment to extract information. Approaches based on pattern recognition are not so sensitive to light artifacts but can only yield a yes/no type of answer when the signal is above or below a certain threshold. Here, we introduce a new method for detecting different concentrations of proteins as well as light artifacts with origami immunosensors and digital logic. The origami design consists of a folded piece of paper with three identical biorecognition sites so that one drop of sample generates three colorimetric signals simultaneously. The three colorimetric signals are then evaluated with an augmented reality app that generates a virtual semaphore that sequentially turns on its green, yellow, and red lights depending on the concentration of analyte. These three Boolean variables pass through "and" and "not" logic gates in a 3-to-8 decoder that enables the semiquantitative detection of proteins and adds a failsafe against erroneous results. The proposed method can detect the model analyte mouse IgG with a limit of detection and sensitivity comparable to densitometry performed under light-controlled conditions. It can also detect the sepsis biomarker procalcitonin at clinically relevant concentrations. With our approach, the detection is performed in real time, and signal processing is not required, which makes it suitable for rapid analyses by nonspecialists at the point of need.

Meeting policy requirements is essential for advancing molecular diagnostic devices from the laboratory to real-world applications and commercialization. Considering policy as a starting point in the design of new technology is a winning strategy. Rapid developments have put mobile biosensors at the frontier of molecular diagnostics, at times outpacing policymakers, and therefore offering new opportunities for breakthroughs in global health. In this Perspective we survey influential global health policies and recent developments in mobile biosensing in order to gain a new perspective for the future of the field. We summarize the main requirements for mobile diagnostics outlined by policy makers such as the World Health Organization (WHO), the World Bank, the European Union (EU), and the Food and Drug Administration (FDA). We then classify current mobile diagnostic technologies according to the manner in which the biosensor interfaces with a smartphone. We observe a trend in reducing hardware components and substituting instruments and laborious data processing steps for user-friendly apps. From this perspective we see software application developers as key collaborators for bridging the gap between policy and practice.

In this manuscript we review the main strategies for detecting changes in intracellular parameters with photoluminescent nanosensors. Examples of the detection of intracellular pH, ion concentration (Na+, Ca2+, Zn2+, Hg2+), reactive oxygen species (ROS, e.g. hydrogen peroxide), variations in temperature, changes in RNA levels and the detection of enzymatic activity are described in detail. The utilisation of nanomaterials such as quantum dots, silicon carbide nanoparticles, nanoscale metal-organic frameworks, upconverting nanoparticles, fluorescent nanoclusters, gold nanoparticles, nanodiamonds, dendrimers and polymeric nanoparticles in bioimaging is highlighted. When compared to molecular probes, nanosensors combine high sensitivity and selectivity with low toxicity, which are crucial parameters for sensing in live cells.

A method is proposed for controlling the number of nanoparticles bound to cell membranes via RGDS peptide-integrin interactions. It consists of propelling nanoparticles bearing the peptides with enzymes (glucose oxidase), which disrupts biomolecular interactions as a function of the concentration of enzyme substrate (glucose).

Biomineralizing organisms can grow nanomaterials with unexpected morphologies in an organic matrix where temporal and vectorial gradients of crystal growth precursors are established. Here, concentration gradients for the crystallization of gold nanoparticles are generated and applied on silicon substrates. Gradients of crystal growth precursors are generated by enzymes patterned as lines that are separated by distances ranging from the micro- to the nanoscale. The concentration of crystallization precursors around the lines separated by nanometric distances is not only determined by mass transport and enzyme activity but also by the nanoscale organization of biocatalysts. This nanoscale organization favors non-classical crystal growth conditions that lead to the formation of nanoparticle clusters containing nanocrystals that are highly crystallographically aligned. The combination of bottom-up crystal growth with top-down electron beam lithography enables the fabrication of micrometric patterns containing gold nanoparticles of different size, shape, and surface density. These are all critical parameters that determine the physical properties of these nanomaterials.

Enzymes are crucial for the cell metabolism and in industrial applications such as the manufacture of biosensors and medicines. A method for inactivating enzymes non-invasively and on demand could reveal the role of a biocatalyst in a particular cell function and enable fine-tuning the therapeutic dose of enzyme-based drugs. In this manuscript we introduce a universal method for inactivating enzymes with light. It consists of attaching the enzymes to photothermal nanoheaters (gold nanorods). Upon irradiation with light resonant with their LSPR the plasmonic nanoparticles generate heat, which denatures the enzymes. The resulting increase in temperature is highest in the vicinity of the nanoheaters, which allows the localized inactivation of nanorod-bound enzymes without damaging other proteins or cell membranes present in the same solution. Here we demonstrate this concept by selectively turning off an enzyme cascade via the selective inactivation of one of its components. We also prove that the target enzyme can be inactivated without unduly damaging cells under controlled irradiation conditions. The results shown here pave the way for using the proposed methodology to discern the role of target enzymes in intracellular signaling pathways.

Enzymes are key components of the bionanotechnology toolbox that possess exceptional biorecognition capabilities and outstanding catalytic properties. When combined with the unique physical properties of nanomaterials, the resulting enzyme-responsive nanoparticles can be designed to perform functions efficiently and with high specificity for the triggering stimulus. This powerful concept has been successfully applied to the fabrication of drug delivery schemes where the tissue of interest is targeted via release of cargo triggered by the biocatalytic action of an enzyme. Moreover, the chemical transformation of the carrier by the enzyme can also generate therapeutic molecules, therefore paving the way to design multimodal nanomedicines with synergistic effects. Dysregulation of enzymatic activity has been observed in a number of severe pathological conditions, and this observation is useful not only to program drug delivery in vivo but also to fabricate ultrasensitive sensors for diagnosing these diseases. In this review, several enzyme-responsive nanomaterials such as polymer-based nanoparticles, liposomes, gold nanoparticles and quantum dots are introduced, and the modulation of their physicochemical properties by enzymatic activity emphasized. When known, toxicological issues related to the utilization nanomaterials are highlighted. Key examples of enzyme-responsive nanomaterials for drug delivery and diagnostics are presented, classified by the type of effector biomolecule, including hydrolases such as proteases, lipases and glycosidases, and oxidoreductases.

We highlight our recent applications of functional peptide nanotubes, self-assembled from short peptides with recognition elements, as building blocks to develop sensors. Peptide nanotubes with high aspect ratios are excellent building blocks for directed assembly into device configurations, and their combining structures with the nanometric diameters and the micrometric lengths enables to bridge the nano-world and the micro-world.

The write stuff: The title method (BCN) is demonstrated for the simultaneous fabrication and patterning of oxide semiconductors at room temperature. BCN involves the production of relatively stable amorphous inks of ZnO, which are nanopatterned with the tip of an atomic force microscope as a pen, which also induces crystallization (see picture). The semiconducting nanostructures could be applied in nanoelectronics and nanooptics applications.