Specific Detection of Proteins by a Nanobody-Functionalized Nanopore Sensor.

Nanopores are label-free single-molecule analytical tools that show great potential for stochastic sensing of proteins. Here, we described a ClyA nanopore functionalized with different nanobodies through a 5-6 nm DNA linker at its periphery. Ty1, 2Rs15d, 2Rb17c, and nb22 nanobodies were employed to specifically recognize the large protein SARS-CoV-2 Spike, a medium-sized HER2 receptor, and the small protein murine urokinase-type plasminogen activator (muPA), respectively. The pores modified with Ty1, 2Rs15d, and 2Rb17c were capable of stochastic sensing of Spike protein and HER2 receptor, respectively, following a model where unbound nanobodies, facilitated by a DNA linker, move inside the nanopore and provoke reversible blockade events, whereas engagement with the large- and medium-sized proteins outside of the pore leads to a reduced dynamic movement of the nanobodies and an increased current through the open pore. Exploiting the multivalent interaction between trimeric Spike protein and multimerized Ty1 nanobodies enabled the detection of picomolar concentrations of Spike protein. In comparison, detection of the smaller muPA proteins follows a different model where muPA, complexing with the nb22, moves into the pore, generating larger blockage signals. Importantly, the components in blood did not affect the sensing performance of the nanobody-functionalized nanopore, which endows the pore with great potential for clinical detection of protein biomarkers.

Hierarchically structural layered double oxides with stretchable nanopores for highly effective removal of protein-bound uremic toxins.

The global outbreak and prevalence of coronavirus disease 2019 (COVID-19) has triggered an urgent demand for family hemodialysis equipment. It is particularly vital to design and apply superior adsorbents to adsorb toxins for reducing the usage of dialysate. In this work, hierarchically structural MgAl layered double oxides (LDO) with stretchable nanopores were exploited through a facile one-pot trisodium citrate (TSC) assistant hydrothermal reaction followed by calcination treatment for effectively adsorbing protein-bound uremic toxins such as hippuric acid (HA) or indoxyl sulfate (IS). The optimized MgAl LDO possessed flower-like spherical morphology, ultrahigh specific surface area (187.3 m2/g) and uniquely stretchable nanopores, which were more conducive to incorporating anions due to their unique memory effect endowing them with promising adsorption capacities for HA or IS. And the adsorption data could be better conformed to pseudo-second-order kinetic model and Langmuir isotherm determining that the maximum adsorption capacity of HA and IS was 129.8 mg/g and 63.1 mg/g, respectively. Furthermore, the computation of molecular size paired with the analysis of adsorption mechanism accurately revealed that high-efficiency toxin capture was mainly attributed to electrostatic interaction for internal intercalation and surface adsorption. Therefore, the application of such delicate LDO as new premium adsorbent would facilitate the development and popularization of family hemodialysis equipment.

RAPID DETECTION OF NOVEL CORONAVIRUS SARS-COV-2 BY SOLID-STATE NANOPORE

The COVID-19 pandemic spreads rapidly and globally. To quell the pandemic propagation, rapid and accurate detection of SARS-CoV-2 is urgently needed. Here, we present a nanopore coupled RT-LAMP method for SARSCoV-2 detection. After comparing all information from the nanopore experiment, we develop a method to use the event rate change of the amplicons translocation event to measure the amplification. As a result, our platform can distinguish positive from negative samples in 15 min with around 65 copies/reaction limit of detection and 100% specificity. We believe that the nanopore coupled RT-LAMP platform would provide a sensitive and specific detection for SARS-COV-2. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Sequence-Specific Recognition of SARS COV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)

The current outbreak of the SARS-CoV-2 caused the COVID-19 disease to spread rapidly globally. Specific and sensitive detection of SARS-CoV-2 is needed to prevent the disease from spread. Here, we present a solid-state CRISPR-Cas12a-assisted nanopores (SCAN) system to detect SARS-CoV-2. We introduced a new scheme using current drops and dwell times of ssDNA reporter translocation events to estimate the cleavage activity. We validated this scheme by a statistical model approximating the reporter length distribution over the cleavage reaction. We believe that the SCAN would provide a sensitive and specific detection method for SARS-COV-2. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

Importance of Sequencing the SARS-CoV-2 Genome Using the Nanopore Technique to Understand Its Origin, Evolution and Development of Possible Cures

The sequencing of the genome of new virus, such as the coronavirus type 2 of the acute severe respiratory syndrome (SARS-CoV-2), is essential and of great importance to mitigate new zoonotic outbreaks, which are caused by mutations present in structural and non-structural proteins that make up the viruses. Sequencing allows tracking the behavior of the virus locally and globally, knowing the route of transmission and spread of the virus, and determine the virulence rate. Current studies have been carried out, using first, second or third generation sequencing techniques, which have allowed reading and analyzing the nucleotides that make up the virus genome. Thus, the benefits of effective technologies to know its genetic composition in the shortest possible time become evident. New technologies are able to monitor an epidemic in real time, monitor the evolution and efficacy of a drug, the development of a vaccine as well as epidemiological advances. This work addresses the Oxford Nanopore sequencing, which is considered the most efficient and applied method for sequencing viruses that cause epidemics. Some of the advantages of using this sequencing are highlighted in this work, such as the ability to perform long readings and be able to obtain sample responses in short time. It’s also able to discover as much information as possible about the pathogen, being an important feature to deal with public health emergencies, such is the case of the COVID-19. © 2022, Springer Nature Switzerland AG.

Emerging Biomedical and Industrial Applications of Nanoporous Materials

Nanoporous materials is a fast-growing subset of nanomaterials with unique intrinsic properties. The advances in fabrication and characterization techniques have enabled scientists to tailor the properties and design a wide range of application specific nanoporous materials. This chapter highlights the key technological advancements that nanoporous materials have achieved in the frontiers of biomedical engineering across analyses, diagnostics and therapeutics. It draws attention to progressive studies like the membrane based organ-on-a-chip (OOC) models of the blood brain barrier and human alveolar that contributed to the advancements in the drug development studies of neurological diseases and COVID-19 to neurochemical biosensing and artificial portable kidneys (Fan et al., ACS Nano 13:8374–8381, 2019;Wang et al., Biotechnol. Bioeng. 114:184–194, 2017;Zhang et al., Adv. Sci. 8:1–14, 2021;Zhou et al., Anal. Chem. 91:3645–3651, 2019). A brief account of the versatile industrial applications of nanoporous materials in chromatography, nanoreactors, energy storage and cutting-edge concepts like nanosized photonic data storage is also illustrated with representative cases. © 2022, Springer Nature Switzerland AG.

Optical trapping assisted label-free and amplification-free detection of SARS-CoV-2 RNAs with an optofluidic nanopore sensor.

Ultrasensitive, versatile sensors for molecular biomarkers are a critical component of disease diagnostics and personalized medicine as the COVID-19 pandemic has revealed in dramatic fashion. Integrated electrical nanopore sensors can fill this need via label-free, direct detection of individual biomolecules, but a fully functional device for clinical sample analysis has yet to be developed. Here, we report amplification-free detection of SARS-CoV-2 RNAs with single molecule sensitivity from clinical nasopharyngeal swab samples on an electro-optofluidic chip. The device relies on optically assisted delivery of target carrying microbeads to the nanopore for single RNA detection after release. A sensing rate enhancement of over 2,000x with favorable scaling towards lower concentrations is demonstrated. The combination of target specificity, chip-scale integration and rapid detection ensures the practicality of this approach for COVID-19 diagnosis over the entire clinically relevant concentration range from 104-109 copies/mL.

Artificially intelligent nanopore for rapid SARS-CoV-2 testing.

An article in Nature Communications reports a method for the rapid detection of SARS-CoV-2 in saliva samples using nanopores and a machine learning algorithm.