Biosensors and Bioelectronics

Printed circuit board (PCB) based biosensors have often utilized hard gold electroplating, that nullifies the cost advantages of this technology as compared to screen printed electrodes. Electroless nickel immersion gold (ENIG) is a popular gold deposition process widely used in PCB manufacturing, but vulnerable to pinhole defects and large surface roughness, which compromises biosensor performance. In this work, we present a method to address these challenges through electrodeposition of methylene blue (MB) to cover surface defects and improve electroactivity of ENIG PCB electrodes. We also demonstrate a process to realize in situ synthesis of gold nanoparticles (AuNPs) using acid-functionalized multi-walled carbon nanotubes (MWCNTs) as scaffold, that are used to immobilize antibody for the target molecule (myeloperoxidase: MPO, early warning biomarker for cardiovascular diseases) through a modified cysteamine/gluteraldehyde based process. The processing steps on the electrode surface are developed in a manner that do not compromise the integrity of the electrode, resulting in repeatable and reliable performance of the sensors. Further, we demonstrate a cost-effective microfluidic packaging process to integrate a capillary pump driven microfluidic channel on the PCB electrode for seamless introduction of samples for testing. We demonstrate the ability of the sensor to distinguish clinically abnormal concentrations of MPO from normal concentrations through extensive characterization using spiked serum and blood plasma samples, with a limit of detection of 0.202 ng/mL.

Rapid diagnosis is critical for the treatment and prevention of diseases. In this research, we report sensing of antibodies specific to SARS-CoV-2 virus in seconds via an electrochemical platform consisting of gold micropillar array electrodes decorated with reduced graphene oxide and functionalized with recombinant viral antigens. The array electrodes are fabricated by Aerosol Jet (AJ) nanoparticle 3D printing, where gold nanoparticles (3-5nm) are assembled in 3D space, sintered, and integrated with a microfluidic device. The device is shown to detect antibodies to SARS-CoV-2 spike S1 protein and its receptor-binding-domain (RBD) at concentrations down to 1pM via electrochemical impedance spectroscopy and read by a smartphone-based user interface. In addition, the sensor can be regenerated within a minute by introducing a low-pH chemistry that elutes the antibodies from the antigens, allowing successive testing of multiple antibody samples using the same sensor. The detection time for the two antibodies tested in this work is 11.5 seconds. S1 protein sensing of its antibodies is specific, which cross-reacts neither with other antibodies nor with proteins such as Nucleocapsid antibody and Interleukin-6 protein. The proposed sensing platform is generic and can also be used for the rapid detection of biomarkers for other infectious agents such as Ebola, HIV, and Zika, which will benefit the public health.

The current COVID-19 global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has become a major public health concern. The ability to identify the viruss presence in infected hosts with sufficient speed and sensitivity is critical to control the epidemic timely. Here, we use self-assembly of arrayed gold nanoparticles (AuNPs) on the coronavirus, which we call the "plasmo-virus particle," to achieve a rapid, sensitive, sample preparation-free assay enabling direct detection of SARS-CoV-2 in a point-of-care (POC) setting. The AuNPs of the plasmo-virus particle serve as plasmonic nanoprobes that specifically bind to the spike protein (S-protein) sites on the surface of SARS-CoV-2. Optical interactions between the self-assembled plasmonic nanoprobes generate multiple modes of highly enhanced plasmonic coupling. Measuring changes of the multimode plasmonic coupling-induced extinction peaks allows for quantifying SARS-CoV-2 at low titers with a limit of detection (LOD) of 1.4 x 101 pfu/mL. Using a miniaturized standalone biochip reading device, we further demonstrate the nano assembly assay for smartphone-operated SARS-CoV-2 detection for viral transport medium (VTM) samples within 10 min without any sample purification steps. We anticipate that the high sensitivity and speed of the POC detection performance of this biosensor technology could be broadly accepted for timely personalized diagnostics of infectious agents under low-resource settings.

Sepsis is a leading cause of mortality worldwide that is difficult to diagnose and manage because this requires simultaneous analysis of multiple biomarkers. Electrochemical detection methods could potentially provide a way to accurately quantify multiple sepsis biomarkers in a multiplexed manner as they have very low limits of detection and require minimal sensor instrumentation; however, affinity-based electrochemical sensors are usually hampered by biological fouling. Here we describe development of an electrochemical detection platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a recently developed nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling. Using nanocomposite coated planar gold electrodes, we constructed a procalcitonin sensor and demonstrated sensitive PCT detection in undiluted serum and clinical samples, as well as excellent correlation with a conventional ELISA (adjusted r2 = 0.95). Sensors for two additional sepsis biomarkers -- C-reactive protein and pathogen-associated molecular patterns -- were developed on the same multiplexed platform and tested in whole blood. Due to the excellent antifouling properties of the nanocomposite coating, all three sensors exhibited specific responses within the clinically significant range without any cross-reactivity in the same channel with low sample volume. This platform enables sensitive simultaneous electrochemical detection of multiple analytes in human whole blood, which can be expanded further to any target analyte with an appropriate antibody pair or capturing probe, and thus, may offer a potentially valuable tool for development of clinical point-of-care diagnostics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/20224683v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1419791org.highwire.dtl.DTLVardef@145c7cdorg.highwire.dtl.DTLVardef@407af3org.highwire.dtl.DTLVardef@1480879_HPS_FORMAT_FIGEXP M_FIG C_FIG

Using the childrens toy, Shrinky-Dink (C), we present an aptamer-based electrochemical (E-AB) assay that recognizes the spike protein of SARS-CoV-2 in saliva for viral infection detection. The low-cost electrodes are implementable at population scale and demonstrate detection down to 0.1 fg mL-1 of the S1 subunit of the spike protein.

The COronaVIrus Disease (COVID-19) is a newly emerging viral disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Rapid increase in the number of COVID-19 cases worldwide led the WHO declare pandemic within a few month after the first case of infection. Due to the lack of a prophylactic measure to control the virus infection and spread, early diagnosis and quarantining of infected as well as the asymptomatic individuals are necessary for the containment of this pandemic. However, the current methods for SARS-CoV-2 diagnosis are expensive and time consuming although some promising and inexpensive technologies are coming out for emergency use. In this work, we report the synthesis of a cheap yet highly sensitive cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical biosensor and its efficacy for rapid detection of spike glycoprotein of SARS-CoV-2 by examining S-RBD protein as the reference material. A simple, low-cost, and one-step electrochemical anodization route was used to synthesize TNTs, followed by an incipient wetting method for cobalt functionalization of the TNTs platform, which is connected to a potentiostat for data collection. This sensor specifically detected the S-RBD protein of SARS-CoV-2 even at very low concentration (range of 14 nM to 1400 nM). Additionally, our sensor showed a linear response in the detection of viral protein with concentration range. In summary, our Co-TNT sensor is highly effective in detecting SARS-CoV-2 S-RBD protein in approximately 30 seconds, which can be explored for developing a point of care diagnostics for rapid detection of SARS-CoV-2 in nasal secretions and saliva samples. AUTHOR SUMMARYSARS-COV-2 is currently a global pandemic on a scale that has not been experienced since the Spanish flu of 1918. One of the reasons why this pandemic virus has spread so quickly is because many infected individuals with SARS-CoV-2 remain asymptomatic and involuntarily transmit the virus before they come down with the symptoms. Therefore, uniform surveillance and quarantining of infected as well as the asymptomatic individuals could provide an effective measure to contain the spread of SARS-CoV-2. However, the current methods for SARS-CoV-2 diagnosis are expensive and time consuming although some inexpensive technologies are getting approvals for emergency use. Our manuscript reports the synthesis of a cheap yet highly sensitive cobalt-functionalized TiO2 nanotubes (Co-TNTs)-based electrochemical biosensor for rapid detection of spike glycoprotein of SARS-CoV-2. Our sensor is synthesized through one-step electrochemical anodization route, followed by an incipient wetting method for cobalt functionalization of TNTs platform. The readout of this sensor is an electrochemical signal collected through a potentiostat, which can be adopted for use through smartphone applications and the development of a point of care diagnostics for COVID-19.

Emerging novel human contagious viruses and pathogens put humans at risk of hospitalization and possibly death due to the unavailability of vaccines and drugs which may take years to develop. Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was classified as a pandemic by theWorld Health Organization and has caused over 550,000 deaths worldwide as of July 2020. Accurate and scalable point-of-care devices would increase screening, diagnosis, and monitoringof COVID-19 patients. Here, we demonstrate rapid label-free electrochemical detection of SARS-CoV-2 antibodies using a commercially available impedance sensing platform. A 16-well plate containing sensing electrodes was pre-coated with receptor binding domain (RBD) of SARS-CoV-2 spike protein, and subsequently tested with samples of anti-SARS-CoV-2 monoclonal antibody CR3022 (0.1 g/ml, 1.0 g/ml, 10 g/ml). Subsequent blinded testing was performed on six serum specimens taken from COVID-19 and non-COVID-19 patients (1:100 dilution factor). The platformwas able to differentiate spikes in impedance measurements from a negative control (1~ milk solution) for all CR3022 samples. Further, successful differentiation and detection of all positive clinical samples from negative control was achieved. Measured impedance values were consistent when compared to standard ELISA test results showing a strong correlation between them (R2 = 0:9). Detection occurs in less than five minutes and the well-based platform provides a simplified and familiar testing interface that can be readily adaptable for use in clinical settings.

The 2019 SARS CoV-2 (COVID-19) pandemic has highlighted the need for rapid and accurate tests to diagnose acute infection and determine immune response to infection. In this work, a multiplexed grating-coupled fluorescent plasmonics (GC-FP) biosensing approach was shown to have 100% selectivity and sensitivity (n = 23) when measuring serum IgG levels against three COVID-19 antigens (spike S1, spike S1S2, and the nucleocapsid protein). The entire biosensing procedure takes less than 30 min, making it highly competitive with well-established ELISA and immunofluorescence assays. GC-FP is quantitative over a large dynamic range, providing a linear response for serum titers ranging from 1:25 to 1:1,600, and shows high correlation with both ELISA and a Luminex-based microsphere immunoassay (MIA) (Pearson r > 0.9). Compatibility testing with dried blood spot samples (n = 63) demonstrated 100% selectivity and 86.7% sensitivity. A machine learning (ML) model was trained to classify dried blood spot samples for prior COVID-19 infection status, based on the combined antibody response to S1, S1S2, and Nuc antigens. The ML model yielded 100% selectivity and 80% sensitivity and demonstrated a higher stringency than a single antibody-antigen response. The biosensor platform is flexible and will readily accommodate detection of multiple immunoglobulin isotypes. Further, it uses sub-nanogram quantities of capture ligand and is thus readily modified to include additional antigens, which is shown by the addition of RBD in later iterations of the test. The combination of rapid, multiplexed, and quantitative detection for both blood serum and dried blood spot samples makes GC-FP an attractive biosensor platform for COVID-19 antibody testing.

Nucleic acids in biofluids are emerging biomarkers for molecular diagnosis of diseases, whose clinical use has been hindered by the lack of sensitive and convenient detection assays. Herein, we report a sensitive nucleic acid detection method based on allosteric DNAzyme biosensors named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a programmable DNAzyme of endonuclease capability. SPOT can be activated once a nucleic acid target of specific sequence binds to its allosteric module to induce conformational reconfiguration of DNAzyme enabling continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive (LOD: femtomolar for miRNAs, attomolar for SARS-CoV-2 RNA), specific (single-nucleotide discrimination), and convenient (one-step, one-pot, preamplification-free) detection of low-abundant nucleic acid biomarkers. For clinical validation, we demonstrated that SPOT is capable of detecting serum miRNAs (e.g., miR-155, miR-21) from patients for the precise diagnosis of breast cancer, gastric cancer, and prostate cancer. Furthermore, SPOT exhibits potent detection capability over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Lastly, SPOT is compatible with point-of-care testing modalities such as lateral flow assay to enable convenient visualization. Hence, we envision that SPOT may serve as a robust platform for sensitive detection of a variety of nucleic acid targets towards clinical applications in molecular diagnosis.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is a highly contagious disease with several variants, continues to spread as part of the global pandemic. With the roll-out of vaccines and development of new therapeutics that may be targeted to distinct viral molecules, there is a need to screen populations for viral antigen-specific SARS-CoV-2 antibodies. Here, we describe a rapid, multiplexed, electrochemical (EC) platform with on-chip control that enables detection of SARS-CoV-2 antibodies in less than 10 min using 1.5 {micro}L of a patient sample. The EC biosensor demonstrated 100% sensitivity and specificity, and an area under the receiver operating characteristic curve of 1, when evaluated using 93 clinical samples, including plasma and dried blood spot samples from 54 SARS-CoV-2 positive and 39 negative patients. This EC biosensor platform enables simple, cost-effective, sensitive, and rapid detection of anti-SARS-CoV-2 antibodies in complex clinical samples, which is convenient for monitoring host humoral responses to vaccination or viral infection in broad population testing, including applications in low-resource settings. We also demonstrate the feasibility of using dried blood spot samples that can be collected locally and transported to distant clinical laboratories at ambient temperature for detection of anti-SARS-CoV-2 antibodies which can be used for serological surveillance and demonstrate the utility of remote sampling.

Infectious diseases such as COVID-19 continue posing significant global health challenges, with recurrent re-infections contributing to long-term symptoms such as cardiac issues and anosmia. Effective management of re-infections relies heavily on maintaining high levels of circulating binding and neutralizing antibodies. Traditional methods for antibody quantification, such as ELISA, face significant challenges, including narrow dynamic ranges and complex sample preparation procedures, which hinder their applications in rapid and routine diagnosis. This study introduces a novel optofluidic biosensing technology, tip optofluidic immunoassay (TOI), that addresses these limitations by enabling the quantitative analysis of binding IgG against multiple SARS-CoV-2 strains from only 1 L of fingertip blood. The proposed TOI system, featuring industrial-grade micro-fabricated immuno-reactors and a portable chemiluminescent imaging station, can provide test results within 12 minutes. For IgG binding assays, TOI possesses a lower limit of detection of 0.1 ng/mL, a dynamic range of 3-4 orders of magnitude, along with a high signal-to-noise ratio (approximately 10,000). This technology not only simplifies the antibody quantification process but also enhances patient compliance and facilitates decentralized testing, which is crucial for infectious disease management. By enabling precise and rapid antibody assessment, this system can support the optimization of vaccination strategies and broader public health responses to COVID-19 and other infectious diseases.

Public healthcare demands effective and pragmatic diagnostic tools to address the escalating challenges in infection management in resource-limited areas. Recent advance in CRISPR-based biosensing promises the development of next-generation tools for disease diagnostics, including point-of-care (POC) testing for infectious diseases. Currently prevailing strategy of developing CRISPR assays exploits only the non-specific trans-cleavage function of a CRISPR-Cas12a/Cas13a system for detection and combines it with an additional pre-amplification reaction to enhance the sensitivity. In contrast to this single-function strategy, here we present a new approach that collaboratively integrates the dual functions of CRISPR-Cas12a: sequence-specific binding and trans-cleavage activity. With this approach, we developed a POC nucleic acid assay termed Solid-Phase Extraction and Enhanced Detection assay Integrated by CRISPR-Cas12a (SPEEDi-CRISPR) that negates the need for preamplification but significantly improves the detection of limit (LOD) from the pM to fM level. Specifically, using Cas12a-coated magnetic beads, this assay combines efficient solid-phase extraction and enrichment of DNA targets enabled by the sequence-specific affinity of CRISPR-Cas12a with the fluorogenic detection by the activated Cas12a on beads. Our proof-of-concept study demonstrated that the SPEEDi-CRISPR assay affords an improved detection sensitivity for human papillomavirus (HPV)-18 with a LOD of 2.3 fM and excellent specificity to discriminate HPV-18 from HPV-16, Parvovirus B19, and scramble HPV-18. Furthermore, this robust assay was readily coupled with a portable smartphone-based fluorescence detector and a lateral flow assay for quantitative detection and visualized readout, respectively. Overall, these results should suggest that our dual-function strategy could pave a new way for developing the next-generation CRISPR diagnostics and that the SPEEDi-CRISPR assay provides a potentially useful tool for point-of-care testing.

Multiplex electronic antigen sensors for detection of SARS-Cov-2 spike glycoproteins or hemagglutinin from Influenza A in liquid samples with characteristics resembling extracted saliva were fabricated using scalable processes with potential for economical mass-production. The sensors utilize the sensitivity and surface chemistry of a two-dimensional MoS2 transducer for attachment of antibody fragments in a conformation favorable for antigen binding. Ultra-thin layers (3 nm) of amorphous MoS2 were directly sputtered over the entire sensor chip at room temperature and laser annealed to create an array of semiconducting 2H-MoS2 active sensor regions between metal contacts. The semiconducting region was functionalized with monoclonal antibody Fab (fragment antigen binding) fragments derived from whole antibodies complementary to either SARS-CoV-2 S1 spike protein or Influenza A hemagglutinin using a papain digestion to cleave the antibodies at the disulfide hinges. The high affinity for the MoS2 transducer surface with some density of sulfur vacancies for the antibody fragment base promoted chemisorption with antigen binding regions oriented for interaction with the sample. The angiostatin converting enzyme 2 (ACE2) receptor protein for the SARS-CoV-2 spike glycoprotein, was tethered to a hexa-histidine (his6) tag at its c-terminus both for purification purposes, as well as a motif for binding to MoS2. This modified protein was also investigated as a bio-recognition element. Electrical resistance measurements of sensors functionalized with antibody fragments and exposed to antigen concentrations ranging from 2-20,000 picograms per milliliter revealed selective responses in the presence of complementary antigens with sensitivity to SARS-CoV-2 or influenza A on the order of pg/mL and comparable to gold-standard diagnostics such as Polymerase Chain Reaction (PCR) analysis. Lack of antigen sensitivity for the larger ACE2 BRE further demonstrates the utility of the engineered antibody fragment/transducer interface in bringing the target antigen closer to the transducer surface for sensitivity required for early detection viral diagnostics.

This study proposes a novel optical method of detecting and reducing SARS-CoV-2 transmission, the virus responsible for the COVID-19 pandemic that is sweeping the world today. SARS-CoV-2 belongs to the {beta}-coronaviruses characterized by the crown-shaped spike protein that protrudes out of the virus particles, giving the virus a "corona" shape; hence the name coronavirus. This virus is similar to the viruses that caused SARS (severe acute respiratory syndrome) and MERS (Middle East respiratory syndrome), the other two coronavirus epidemics that were recently contained within the last ten years. The technique being proposed uses a light source from a smart phone and a mobile spectrophotometer to enable detection of viral proteins in solution or paper as well as protein-protein interactions. The proof-of-concept is shown by detecting soluble preparations of spike protein subunits from SARS-CoV-2, followed by detection of the actual binding potential of the spike protein with its host receptor, the angiotensin-converting enzyme 2 (ACE2). The results are validated by showing that this method can detect antigen-antibody binding using two independent viral protein-antibody pairs. The binding could be detected optically both in solution and on a solid support such as nitrocellulose membrane. Finally, this technique is combined with DC bias to show that introduction of a current into the system can be used to disrupt the antigen-antibody reaction, suggesting that the proposed extended technique can be a potential means of not only detecting the virus, but also reducing virus transmission by disrupting virus-receptor interactions electrically. SignificanceThe measured intensity of light can reveal information about different cellular parameters under study. When light passes through a bio-composition, the intensity is associated with its content. The nuclei size, cell shape and the refractive index variation of cells contributes to light intensity. In this work, an optical label-free real time detection method incorporating the smartphone light source and a portable mini spectrometer for SARS-CoV-2 detection was developed based on the ability of its spike protein to interact with the ACE2 receptor. The light interactions with control and viral protein solutions were capable of providing a quick decision regarding whether the sample under test was positive or negative, thus enabling SARS-CoV-2 detection in a rapid manner.

The SARS-CoV-2 virus has spread globally causing coronavirus disease 2019 (COVID-19). Rapidly and accurately identifying viral infections is an ongoing necessity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to produce a DNA allonamer with two distinct binding domains made allosteric through a linker section; one domain binds SARS-CoV-2 spike (S) protein, inducing a conformational change that allows the reporter domain to bind a fluorescent reporter molecule. We used bead-based fluorescence and immunofluorescence assays to confirm the allonamers affinity and specificity for S-protein and confirmed that the allonamer can bind to S-proteins with mutations corresponding to those of the alpha, beta, gamma, and delta variants. We then developed the allonamer-based Quantum-Logic Aptamer Analyte Detection (Q-LAAD) test, a rapid, high-throughput antigen test for qualitative detection of SARS-CoV-2 in clinical settings. We validated Q-LAAD against retrospective and prospective clinical anterior nasal swab samples collected from symptomatic patients suspected of having COVID-19. Q-LAAD showed 97% sensitivity and 100% specificity compared to the RT-qPCR assay. Q-LAAD has a limit of detection (LOD) of 1.88 TCID50/mL, is cost-effective and convenient, and requires only a common fluorescence plate reader. Q-LAAD may be a useful clinical diagnostic tool in the fight against SARS-CoV-2. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/22280297v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@1250f56org.highwire.dtl.DTLVardef@11eb2dorg.highwire.dtl.DTLVardef@6711dcorg.highwire.dtl.DTLVardef@c5bf3e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIAllonamers are allosterically-regulated DNA aptamers with multiple binding pockets C_LIO_LIQ-LAAD uses allonamers to detect SARS-CoV-2 spike protein in clinical samples C_LIO_LIQ-LAAD has high sensitivity and specificity and a low limit of detection C_LIO_LIQ-LAAD can detect spike proteins from multiple SARS-CoV-2 variants C_LIO_LIQ-LAAD is a dynamic, cost-effective rapid antigen test for detection of SARS-CoV-2 C_LI

Multiplexed solid-phase polymerase chain reaction (SP-PCR) has emerged as an indispensable modality for concurrent amplification of multiple genetic loci within a singular reaction vessel, facilitating efficient molecular diagnostics. Nevertheless, SP-PCR has seldom been integrated into point-of-care diagnostic devices due to several technical challenges, such as bubble formation during PCR, long reaction time and low fluorescence signals generated from the PCR products on a solid surface. To circumvent these constraints, we engineered a microfluidic chip comprising SP-PCR and nanophotonic enhancement to enable highly sensitive, high-throughput, and cost-efficient molecular diagnostics. The chips vertical orientation integrates preloaded reagent chambers for sequential lysis, washing, elution, and amplification, driven by a synchronized stepper motor and air vacuum, achieving robust nucleic acid purification and reverse transcription-PCR and enables bubble-free, gravity-assisted fluid dynamics during the PCR thermocycling. Thermal cycling is expedited through a dual-heater configuration alternating at sub-second intervals, obviating active cooling and shortening the reaction time. All-dielectric nanostructured metasurface was incorporated beneath the PCR chamber, allowing for the facile immobilization of DNA arrays to conduct SP-PCR. Taking advantage of guided-mode resonance supported by the metasurface and the SP-PCR approaches permits multiplexed detection and achieves a detection limit of 10 copies/reaction for SARS-CoV-2, highlighting the platforms potential for point-of-care diagnostics, personalized medicine, and high-throughput pathogen surveillance. Facile fabrication and automation emphasize scalability for mass production and deployment and collectively represent advancement in point-of-care diagnostics.

Digital assays are in wide development for biomarker quantification at the single-molecule level, but the common use of surface-pulldown steps limits both analytical sensitivity and throughput. Here, we develop surface-free, wash-free, in-solution assays with a sensitivity slope approaching unity for sequence-specific counting of microRNAs (miRs) relevant to metastatic castration-resistant prostate cancer (mCRPC). These assays are enabled by DNA nanoflowers (DNFs) densely encoded with [~]200 fluorescent quantum dots (QDs) that assemble in situ stoichiometrically to miRs. The QD-DNFs are detected as single events in solution by fluorescence microscopy or flow cytometry without washing away unbound labels. A [~]50 aM limit of detection and high agreement with absolute target count (0.95) were achieved by machine learning-guided assay optimization, providing the potential for calibration-free measurements. Multiple miR sequences could be distinguished through ratiometric and colorimetric (5-color) QD signatures with a single excitation source for flexible detection scenarios in static solution or flow streams. The assays were applied for detecting exosomal miRs from small-volume plasma of mCRPC patients and showed strong agreement with RT-qPCR, but with more reliable detection of the trace prognostic biomarker miR-375. Consistent with our prior reports using large volume blood draws, higher plasma levels of miR-375 were associated with poor survival of patients with mCRPC. We anticipate that in-solution absolute counting of clinical biomarkers in plasma will enable robust molecular analysis of trace biomarkers needed for the translation of cancer precision medicine.

Cardiovascular disease (CVD) is a serious worldwide health concern that necessitates the development of novel diagnostic techniques for early identification and personalized healthcare management. Even before the insights provided by gut microbiota, current research has demonstrated the importance of circulating microbiome (CMB) in the evolution of cardiometabolic illness risk and progression. We developed a nanobiosensor that uses specific labeled capture probes with perovskite quantum dots (PQDs) to detect the targeted 16S rRNA sequences in the peripheral milieu. With ideal applicability, specificity, and sensitivity, this sensor delivers unique insights into the presence and characterization of circulating microbiota signatures. Developing a nanophotonic microbiome detection method in body fluids may pave the way for creating a distinctive tool for CVD risk prediction for population-based screening programs in low and middle-income countries.

Over the last 6 years, five-year survival rate for pancreatic cancer patients has increased from 6 to 10% after the initial diagnosis, which makes it one of the deadliest cancer types. This disease is known as the "silent killer" because early detection is challenging due to the location of the pancreas in the body and the nonspecific clinical symptoms. The Bossmann group has developed ultrasensitive nanobiosensors for protease/arginase detection comprised of Fe/Fe3O4 nanoparticles, cyanine 5.5, and designer peptide sequences linked to TCPP. Initial data obtained from both gene expression analysis and protease/arginase activity detection in serum indicated the feasibility of early pancreatic cancer detection. Several matrix metalloproteinases (MMPs, -1, -3, and -9), cathepsins (CTS) B and E, neutrophil elastase, and urokinase plaminogen activator (uPA) have been identified as candidates for proximal biomarkers. In this study, we have confirmed our initial results from 2018 performing serum sample analysis assays using a larger group sample size (n=159), which included localized (n=33) and metastatic pancreatic cancer (n=50), pancreatitis (n=26), and an age-matched healthy control group (n=50). The data obtained from the eight nanobiosensors capable of ultrasensitive protease and arginase activity measurements were analyzed by means of an optimized information fusion-based hierarchical decision structure. This permits the modeling of early-stage detection of pancreatic cancer as a multi-class classification problem. The most striking result is that this methodology permits the detection of localized pancreatic cancers from serum analyses with 96% accuracy.

The isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics.