Artificial Intelligence-Assisted Multiparameter Size Discrimination of Silver Nanoparticles through Electrochemical Collision.

Single particle collision is an important tool for size analysis at the individual particle level; however, due to complex dynamic behaviors of nanoparticles on the surface of an electrode, the accuracy of size discrimination is limited. A silver (Ag) nanoparticle (NP) was chosen as the research target, and the dynamic behavior of Ag NPs was simplified by enhancing adsorption between Ag NP and Au ultramicroelectrode (UME) in alkaline media. Immediately after, accurate dynamic and thermodynamic information on single Ag NP was accurately extracted from collision events, including current intensity, transferred charge, and duration time. On the basis that there were differences between parameters of different-sized Ag NPs, multiparameter size discrimination was proposed, which improved the accuracy compared to single-parameter discrimination. More intriguingly, multiparameter analysis was combined with artificial intelligence, a tool adept at processing multidimensional data, for the first time. Finally, artificial intelligence-assisted multiparameter size discrimination was successfully used to intelligently distinguish mixed Ag NPs, with an optimal accuracy of more than 95%. To sum up, the artificial intelligence-assisted multiparameter method showed an excellent ability to quickly achieve the most accurate size discrimination of nanoparticles at the level of individual particle and provide an effective guidance for the application of nanoparticles.

Current Lifetime of Single-Nanoparticle Electrochemical Collision for In Situ Monitoring Nanoparticles Agglomeration and Aggregation.

In situ monitoring of the agglomeration/aggregation process of nanoparticles (NPs) is crucial because it seriously affects cell entry, biosafety, catalytic performance of NPs, and so on. Nevertheless, it remains hard to monitor the solution phase agglomeration/aggregation of NPs via conventional techniques such as electron microscopy, which requires sample pretreatment and cannot represent native state NPs in solution. Considering that single-nanoparticle electrochemical collision (SNEC) is powerful to detect NPs in solution at the single-particle level, and the current lifetime, which refers to the time that current intensity decays to 1/e of the original value, is skilled in distinguishing different sized NPs, herein, a current lifetime-based SNEC has been developed to distinguish a single Au NP (d = 18 nm) from its agglomeration/aggregation. Based on this, the agglomeration/aggregation process of small-sized NPs and the discrimination of agglomeration vs aggregation have been carefully investigated at the single-particle level. Results showed that the agglomeration/aggregation of Au NPs (d = 18 nm) in 0.8 mM HClO4 climbed from 19% to 69% over two hours, whereas there was no visible granular sediment, and Au NPs tended to agglomerate rather than aggregate irreversibly under normal conditions. Hence, the proposed current lifetime-based SNEC could serve as a complementary method to in situ monitor the agglomeration/aggregation of small-sized NPs in solution at the single-particle level and provide effective guidance for the practical application of NPs.

Single-Nanoparticle Collision Electrochemistry Biosensor Based on an Electrocatalytic Strategy for Highly Sensitive and Specific Detection of H7N9 Avian Influenza Virus.

Single-nanoparticle collision electrochemistry (SNCE) has gradually become an attractive analytical method due to its advantages in analytical detection, such as a fast response, low cost, low sample consumption, and in situ real-time detection of analytes. However, the biological analyte's direct detection based on the SNCE blocking mode has the problems of low sensitivity and specificity. In this work, an SNCE biosensor based on SNCE electrocatalytic strategy was used for the detection of H7N9 AIV. Nucleic acid aptamers were introduced to recognize the target virus (H7N9 AIV). After the recognition event, ssDNA1 was released and hybridized with another ssDNA2. Owing to the nicking endonuclease Nt.AlwI-mediated target nucleic acid cyclic amplification, one virus particle can indirectly induce the release of 4.2 × 106 Au NPs that can be counted by the SNCE electrocatalytic strategy. The high conversion efficiency greatly improved the detection sensitivity, and the detection limit was as low as 24.3 fg/mL. Therefore, the constructed biosensor can achieve a highly sensitive and specific detection of H7N9 AIV and show a great potential in bioanalytical application.

Current Lifetime of Single-Nanoparticle Collision for Sizing Nanoparticles.

Accurate size analysis of nanoparticles (NPs) is vital for nanotechnology. However, this cannot be realized based on conventional single-nanoparticle collision (SNC) because the current intensity, a thermodynamic parameter of SNC for sizing NPs, is always smaller than the theoretical value due to the effect of NP movements on the electrode surface. Herein, a size-dependent dynamic parameter of SNC, current lifetime, which refers to the time that the current intensity decays to 1/e of the original value, was originally utilized to distinguish differently sized NPs. Results showed that the current lifetime increased with NP size. After taking the current lifetime into account rather than the current intensity, the overlap rates for the peak-type current transients of differently sized Pt NPs (10 and 15 nm) and Au NPs (18 and 35 nm) reduced from 73 and 7% to 45 and 0%, respectively, which were closer to the theoretical values (29 and 0%). Hence, the proposed SNC dynamics-based method holds great potential for developing reliable electrochemical approaches to evaluate NP sizes accurately.

Size-Resolved Single Entity Collision Biosensing for Dual Quantification of MicroRNAs in a Single Run.

Limited to the accuracy of size resolution, single entity collision biosensing (SECBS) for multiplex immunoassays remains challenging, because it is difficult to get the true value of nanoparticle (NP) sizes based on the current intensity due to the complex movement of NPs on the electrode surface. Considering that the size-dependent movement of NPs meanwhile will generate a characteristic current shape, in this work, the huge difference in the current rise time of 5 and 15 nm Pt NPs colliding on an Au ultramicroelectrode (d = 30 µm) was originally used to develop a size-resolved SECBS for multiplex immunoassays of miRNAs. The limit concentration that can be detected was 0.5 fM. Compared with conventional electrochemical biosensors for multiplex immunoassays, for the size-resolved SECBS, one does not need to worry about potential overlapping. Therefore, the proposed method demonstrates a promising potential for the application of SECBS in multiplex immunoassays.

Ultrasensitive Electrochemiluminescence Biosensor Based on Closed Bipolar Electrode for Alkaline Phosphatase Detection in Single Liver Cancer Cell.

An ultrasensitive electrochemiluminescence (ECL) biosensor was proposed based on a closed bipolar electrode (BPE) for the detection of alkaline phosphatase (ALP). For most of the BPE-ECL biosensors, an effective signal amplification strategy was the key to enhance the sensitivity of the system. Herein, the signal amplification strategy of the enzyme catalysis was utilized in the BPE-ECL system. Au nanoparticles (NPs) were electrodeposited on the cathode surface of the ITO electrode to improve the stability and sensitivity of the signal. Compared with the previous BPE-ECL biosensors, the sensitivity was increased by at least 3 orders of magnitude. The biosensor showed high sensitivity and specificity of ALP detection with a detection limit of as low as 3.7 aM. Besides, it was further applied to the detection of ALP in different types of cells and successfully realized ALP detection in single Hep G2 cell, which had a huge application prospect in single biomolecule detection or single cell analysis.

One-to-Many Single Entity Electrochemistry Biosensing for Ultrasensitive Detection of microRNA.

Single-entity electrochemistry (SEEC), a promising method for biosensing, has an intrinsic limitation on sensitivity since at most one colliding entity can be successfully triggered by one target. Here, we take advantage of one-to-many (1:n) signal amplification to develop a new single-entity electrochemistry biosensing (SEECBS), integrating satellite magnetic nanoparticle (MN)-DNA-Pt nanoparticle (NP) conjugates, duplex-specific nuclease (DSN) assisted Pt NPs releasing with stabilization, and effective collision of small sized and nearly naked Pt NPs. Compared with conventional SEECBS, the 1:n SEECBS can successfully enrich ∼2 nM Pt NPs by adding 50 aM microRNA (miRNA), in other words, ∼4 × 107 Pt NPs can be triggered by one target. The proposed SEECBS allows the detection of 47 aM miRNA-21, nearly 6 orders of magnitude lower than the previous work, and discrimination of nontarget miRNAs containing even single-nucleotide mismatch. Besides, this method has also been successfully demonstrated for quantification of miRNA in different cell lines. Therefore, the proposed method holds great potential for the application of SEECBS in early diagnosis and prognosis monitoring of cancer.

Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian Influenza Virus Detection Based on Fluorescent Magnetic Multifunctional Nanospheres.

The fluorescence method has made great progress in the construction of sensitive sensors but the background fluorescence of the matrix and photobleaching limit its broad application in clinical diagnosis. Here, we propose a digital single virus immunoassay for multiplex virus detection by using fluorescent magnetic multifunctional nanospheres as both capture carriers and signal labels. The superparamagnetism and strong magnetic response ability of nanospheres can realize efficient capture and separation of targets without sample pretreatment. Due to their distinguishable fluorescence imaging and photostability, the nanospheres enable single-particle counting for ultrasensitive multiplexed detection. Furthermore, the integration of digital analysis provided a reliable quantitative strategy for the detection of rare targets. Based on multifunctional nanospheres and digital analysis, a digital single virus immunoassay was proposed for simultaneous detection of H9N2, H1N1, and H7N9 avian influenza virus without complex signal amplification, whose detection limits were 0.02 pg/mL. Owing to its good specificity and anti-interference ability, the method showed great potential in single biomolecules, multiplexed detection, and early diagnosis of diseases.

Digital Single Virus Electrochemical Enzyme-Linked Immunoassay for Ultrasensitive H7N9 Avian Influenza Virus Counting.

Electrochemistry has been widely used to explore fundamental properties of single molecules due to its fast response and high specificity. However, the lack of efficient signal amplification strategies and quantitative method limit its clinical application. Here, we proposed a digital single virus electrochemical enzyme-linked immunoassay (digital ELISA) for H7N9 avian influenza virus (H7N9 AIV) counting by integration of digital analysis, bifunctional fluorescence magnetic nanospheres (bi-FMNs) with monolayer gold nanoparticles (Au NPs) modified microelectrode array (MA). Bi-FMNs are fabricated by coimmobilizing polyclonal antibody (pAb) and alkaline phosphatase (ALP). At most, one target will be captured per bi-FMNs by controlling the proportion of bi-FMNs to target concentrations (≥5:1). The introduction of digital analysis can solve signal fluctuation and the reliability of single virus detection, enabling the digital ELISA to be sensitively and accurately applied for H7N9 AIV detection with a low detection limit of 7.8 fg/mL, which is greatly promising in single biomolecular detection, early diagnosis of disease, and practical application.