Integration of Metal-Organic Polyhedra onto a Nanophotonic Sensor for Real-Time Detection of Nitrogenous Organic Pollutants in Water.

The grave health and environmental consequences of water pollution demand new tools, including new sensing technologies, for the immediate detection of contaminants in situ. Herein, we report the integration of metal-organic cages or polyhedra (MOCs/MOPs) within a nanophotonic sensor for the rapid, direct, and real-time detection of small (<500 Da) pollutant molecules in water. The sensor, a bimodal waveguide silicon interferometer incorporating Rh(II)-based MOPs as specific chemical receptors, does not require sample pretreatment and enables minimal expenditure of time and reagents. We validated our sensor for the detection of two common pollutants: the industrial corrosion inhibitor 1,2,3-benzotriazole (BTA) and the systemic insecticide imidacloprid (IMD). The sensor offers a fast time-to-result response (15 min), high sensitivity, and high accuracy. The limit of detection (LOD) in tap water for BTA is 0.068 µg/mL and for IMD, 0.107 µg/mL, both of which are below the corresponding toxicity thresholds defined by the European Chemicals Agency (ECHA). By combining innovative chemical molecular receptors such as MOPs with state-of-the-art photonic sensing technologies, our research opens the path to implement competitive sensor devices for in situ environmental monitoring.

Using Electrochemical Immunoassay in a Novel Microtiter Plate to Detect Surface Markers of Preeclampsia on Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.

One-Step and Real-Time Detection of microRNA-21 in Human Samples for Lung Cancer Biosensing Diagnosis.

The rapid diagnosis of cancer, especially in its early stages, is crucial for on-time medical treatment and for increasing the patient survival rate. Lung cancer shows the highest mortality rate and the lowest 5-year survival rate due to the late diagnosis in advanced cancer stages. Providing rapid and reliable diagnostic tools is a top priority to address the problem of a delayed cancer diagnosis. We introduce a nanophotonic biosensor for the direct and real-time detection in human plasma of the microRNA-21-5p biomarker related to lung cancer. The biosensor employs a silicon photonic bimodal interferometric waveguide that provides a highly sensitive detection in a label-free format. We demonstrate a very competitive detectability for direct microRNA-21-5p biomarker assays in human plasma samples (estimated LOD: 25 pM). The diagnostic capability of our biosensor was validated by analyzing 40 clinical samples from healthy individuals and lung cancer patients, previously analyzed by reverse-transcription quantitative polymerase chain reaction (qRT-PCR). We could successfully identify and quantify the levels of microRNA in a one-step assay, without the need for DNA extraction or amplification steps. The study confirmed the significance of implementing this biosensor technique compared to the benchmarking molecular analysis and showed excellent agreement with previous results employing the traditional qRT-PCR. This work opens new possibilities for the true implementation of point-of-care biosensors that enable fast, simple, and efficient early diagnosis of cancer diseases.

Coating Bioactive Microcapsules with Tannic Acid Enhances the Phenotype of the Encapsulated Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) may be differentiated into any adult cell type and therefore hold incredible promise for cell therapeutics and disease modeling. There is increasing interest in three-dimensional (3D) hPSC culture because of improved differentiation outcomes and potential for scale up. Our team has recently described bioactive heparin (Hep)-containing core-shell microcapsules that promote rapid aggregation of stem cells into spheroids and may also be loaded with growth factors for the local and sustained delivery to the encapsulated cells. In this study, we explored the possibility of further modulating bioactivity of microcapsules through the use of an ultrathin coating composed of tannic acid (TA). Deposition of the TA film onto model substrates functionalized with Hep and poly(ethylene glycol) was characterized by ellipsometry and atomic force microscopy. Furthermore, the presence of the TA coating was observed to increase the amount of basic fibroblast growth factor (bFGF) incorporation by up to twofold and to extend its release from 5 to 7 days. Most significantly, TA-microcapsules loaded with bFGF induced higher levels of pluripotency expression compared to uncoated microcapsules containing bFGF. Engineered microcapsules described here represent a new stem cell culture approach that enables 3D cultivation and relies on local delivery of inductive cues.

Label-Free Plasmonic Biosensor for Rapid, Quantitative, and Highly Sensitive COVID-19 Serology: Implementation and Clinical Validation.

Serological tests are essential for the control and management of COVID-19 pandemic (diagnostics and surveillance, and epidemiological and immunity studies). We introduce a direct serological biosensor assay employing proprietary technology based on plasmonics, which offers rapid (<15 min) identification and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in clinical samples, without signal amplification. The portable plasmonic device employs a custom-designed multiantigen (RBD peptide and N protein) sensor biochip and reaches detection limits in the low ng mL-1 range employing polyclonal antibodies. It has also been implemented employing the WHO-approved anti-SARS-CoV-2 immunoglobulin standard. A clinical validation with COVID-19 positive and negative samples (n = 120) demonstrates its excellent diagnostic sensitivity (99%) and specificity (100%). This positions our biosensor as an accurate and easy-to-use diagnostics tool for rapid and reliable COVID-19 serology to be employed both at laboratory and decentralized settings for the disease management and for the evaluation of immunological status during vaccination or treatment.

Fast and Accurate Pneumocystis Pneumonia Diagnosis in Human Samples Using a Label-Free Plasmonic Biosensor.

Pneumocystis jirovecii is a fungus responsible for human Pneumocystis pneumonia, one of the most severe infections encountered in immunodepressed individuals. The diagnosis of Pneumocystis pneumonia continues to be challenging due to the absence of specific symptoms in infected patients. Moreover, the standard diagnostic method employed for its diagnosis involves mainly PCR-based techniques, which besides being highly specific and sensitive, require specialized personnel and equipment and are time-consuming. Our aim is to demonstrate an optical biosensor methodology based on surface plasmon resonance to perform such diagnostics in an efficient and decentralized scheme. The biosensor methodology employs poly-purine reverse-Hoogsteen hairpin probes for the detection of the mitochondrial large subunit ribosomal RNA (mtLSU rRNA) gene, related to P. jirovecii detection. The biosensor device performs a real-time and label-free identification of the mtLSU rRNA gene with excellent selectivity and reproducibility, achieving limits of detection of around 2.11 nM. A preliminary evaluation of clinical samples showed rapid, label-free and specific identification of P. jirovecii in human lung fluids such as bronchoalveolar lavages or nasopharyngeal aspirates. These results offer a door for the future deployment of a sensitive diagnostic tool for fast, direct and selective detection of Pneumocystis pneumonia disease.

Optical nanogap antennas as plasmonic biosensors for the detection of miRNA biomarkers.

Nanoplasmonic biosensors based on nanogap antenna structures usually demand complex and expensive fabrication processes in order to achieve a good performance and sensitive detection. We here report the fabrication of large-area nanoplasmonic sensor chips based on nanogap antennas by employing a customized, simple and low-cost colloidal lithography process. By precisely controlling the angle for tilted e-beam metal evaporation, an elliptical mask is produced, which defines the total length of the dipole antenna nanostructures while assuring that the plasmonic response is oriented in the same direction along the sensor chip. Large-area sensor chips of nanogap antennas formed by pairs of gold nanodisks separated by gaps with an average size of 11.6 ± 4.7 nm are obtained. The optical characterization of the nanogap antenna structures in an attenuated total reflection (ATR) configuration shows a bulk refractive index sensitivity of 422 nm per RIU, which is in agreement with FDTD numerical simulations. The biosensing potential of the cm2-sized nanostructured plasmonic sensor chips has been evaluated for the detection of miRNA-210, a relevant biomarker for lung cancer diagnosis, through a DNA/miRNA hybridization assay. A limit of detection (LOD) of 0.78 nM (5.1 ng mL-1) was achieved with no need of further amplification steps, demonstrating the high sensitivity of these plasmonic nanogap antennas for the direct and label-free detection of low molecular weight biomolecules such as miRNAs.

Advanced Evanescent-Wave Optical Biosensors for the Detection of Nucleic Acids: An Analytic Perspective.

Evanescent-wave optical biosensors have become an attractive alternative for the screening of nucleic acids in the clinical context. They possess highly sensitive transducers able to perform detection of a wide range of nucleic acid-based biomarkers without the need of any label or marker. These optical biosensor platforms are very versatile, allowing the incorporation of an almost limitless range of biorecognition probes precisely and robustly adhered to the sensor surface by covalent surface chemistry approaches. In addition, their application can be further enhanced by their combination with different processes, thanks to their integration with complex and automated microfluidic systems, facilitating the development of multiplexed and user-friendly platforms. The objective of this work is to provide a comprehensive synopsis of cutting-edge analytical strategies based on these label-free optical biosensors able to deal with the drawbacks related to DNA and RNA detection, from single point mutations assays and epigenetic alterations, to bacterial infections. Several plasmonic and silicon photonic-based biosensors are described together with their most recent applications in this area. We also identify and analyse the main challenges faced when attempting to harness this technology and how several innovative approaches introduced in the last years manage those issues, including the use of new biorecognition probes, surface functionalization approaches, signal amplification and enhancement strategies, as well as, sophisticated microfluidic solutions.

Label-Free Nanoplasmonic Biosensing of Cancer Biomarkers for Clinical Diagnosis.

Biosensing of cancer biomarkers enabling early diagnosis of cancer constitutes an essential tool for clinical intervention and application of novel therapies against cancer disease. Optical biosensor instruments as point-of-care (POC) devices and operating under label-free scheme have demonstrated to provide fast, simple, and high-sensitivity assays even at home care environment. Nanoplasmonic biosensors are thought to be a powerful tool for detection of complex analytes of relevant clinical applications. Using high-throughput fabrication techniques, large surface patterned with gold nanodisk structures is obtained showing surface sensitivities with limit of detection (LOD) in the order of picomolar concentration range. Here, we describe two major assay methodologies used for detection of lung and colorectal cancer, respectively. Particularly, we have selected a complementary hybridization DNA/RNA assay for the assessment of two miRNAs (miRNA-210 and miRNA-205) for detection of lung cancer. However, for colorectal cancer we present the detection of four tumor-associated antigen (TAA) biomarkers (MAPKAPK3, PIM-1, STK4, and GTF2B) as possible TAA targets for autoantibody production. Strategies for detecting these biomarkers in real samples such as serum are also presented, demonstrating the capabilities of these assays to be transferred to real clinical settings.

Early sepsis diagnosis via protein and miRNA biomarkers using a novel point-of-care photonic biosensor.

Sepsis is a condition characterized by a severe stage of blood-infection often leading to tissue damage, organ failure and finally death. Fast diagnosis and identification of the sepsis stage (sepsis, severe sepsis or septic shock) is critical for the patient's evolution and could help in defining the most adequate treatment in order to reduce its mortality. The combined detection of several biomarkers in a timely, specific and simultaneous way could ensure a more accurate diagnosis. We have designed a new optical point-of-care (POC) device based on a phase-sensitive interferometric biosensor with a label-free microarray configuration for potential high-throughput evaluation of specific sepsis biomarkers. The sensor chip, which relies on the use of metallic nanostructures, provides versatility in terms of biofunctionalization, allowing the efficient immobilization of different kind of receptors such as antibodies or oligonucleotides. We have focused on two structurally different types of biomarkers: proteins, including C-reactive protein (CRP) and Interleukin 6 (IL6), and miRNAs, using miRNA-16 as an example. Limits of Detection (LoD) of 18 µg mL-1, 88 µg mL-1 and 1 µM (6 µg mL-1) have been respectively obtained for CRP, IL6 and miRNA-16 in individual assays, with high accuracy and reproducibility. The multiplexing capabilities have also been assessed with the simultaneous analysis of both protein biomarkers.

Label-free Bacteria Quantification in Blood Plasma by a Bioprinted Microarray Based Interferometric Point-of-Care Device.

Existing clinical methods for bacteria detection lack speed, sensitivity, and, importantly, point-of-care (PoC) applicability. Thus, finding ways to push the sensitivity of clinical PoC biosensing technologies is crucial. Here we report a portable PoC device based on lens-free interferometric microscopy (LIM). The device employs high performance nanoplasmonics and custom bioprinted microarrays and is capable of direct label-free bacteria ( E. coli) quantification. With only one-step sample handling we offer a sample-to-data turnaround time of 40 min. Our technology features detection sensitivity of a single bacterial cell both in buffer and in diluted blood plasma and is intrinsically limited by the number of cells present in the detection volume. When employed in a hospital setting, the device has enabled accurate categorization of sepsis patients (infectious SIRS) from control groups (healthy individuals and noninfectious SIRS patients) without false positives/negatives. User-friendly on-site bacterial clinical diagnosis can thus become a reality.