Amplification-Free Digital Immunoassay down to the Attomolar Level by Synergistic Sedimentation of Brownian Motion Suppression and Dehydration Transfer.

Amplification-free digital immunoassays (DIAs) typically utilize optical nanoparticles to enhance single immunocomplex molecule detection. The efficiency and uniformity of transferring the nanoparticles from a bulk solution to a solid surface determine the limit of detection (LOD) and the accuracy of DIAs. Previous methods suffer from issues like low efficiency, nonuniform distribution, and particle aggregation. Here, we present a novel technique named synergistic sedimentation of Brownian motion suppression and dehydration transfer (SynSed) for nanoparticles using water-soluble polymers. The efficiency of transferring quantum dots (QDs) was increased from 10.7 to 91.4%, and the variation in QD distribution was restricted to 8.8%. By incorporating SynSed into DIAs, we achieved a remarkable reduction in the LOD (down to 3.9 aM) for carcinoembryonic antigen and expanded the dynamic range to cover 3 orders of magnitude in concentration, ranging from 0.01 to 10 fM. DIAs enhanced with SynSed possess ultrahigh sensitivity, advanced accuracy, and specificity, offering a great premise in early disease diagnostics, risk stratification, and treatment response monitoring.

Homogeneous immunoassay utilizing fluorescence resonance energy transfer from quantum dots to tyramide dyes deposited on full immunocomplexes.

There is an urgent need for homogeneous immunoassays that offer sufficient sensitivity for routine clinical practice. In this study, we have developed a highly sensitive, fluorescence resonance energy transfer (FRET)-based homogeneous immunoassay. Unlike previous FRET-based homogeneous immunoassays, where acceptors were attached to antibody molecules located far from the donor, we employed acceptors to label the entire sandwich-structured immunocomplex, including two antibodies and one antigen. As a result, the FRET signal was amplified by a factor of 10, owing to the reduced distance between the donor and acceptors. We validated our method by quantifying carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) in PBS buffer and blank plasma. The limits of detection (LOD) for CEA and AFP in both PBS buffer and blank plasma were comparable, reaching sub-femtomolar levels. Furthermore, we successfully quantified CEA and AFP in three human plasma samples, thereby confirming the reliability of our method for clinical applications.

Multiplexed Homogeneous Immunoassay Based on Counting Single Immunocomplexes together with Dark-Field and Fluorescence Microscopy.

The development of multiplexed immunoassays is impeded by the difficulty in distinguishing labeled immunocomplexes from free probes and nonspecifically bound probes. Here, we attempted to overcome this issue by counting core-satellite-structured immunocomplexes simultaneously using dark-field and fluorescence microscopy. The tumor biomarkers of carcinoembryonic antigen (CEA), α-fetoprotein (AFP), and prostate-specific antigen (PSA) were chosen as model targets. Gold nanoparticles (AuNPs) with diameters of 70 nm were coated with the detection antibodies of the three targets. Quantum dot (QD) 525, QD 585, and QD 655 were modified with the capture antibodies of CEA, AFP, and PSA, respectively. Then, an immunocomplex containing one AuNP and one or several QDs was formed, whereas free and nonspecifically bound probes had either one AuNP or one QD. When observed with a transmission grating-based spectral microscope, the immunocomplexes had overlapping scattering and fluorescent spectral images and were therefore identified and quantified precisely. The biomarkers inside the immunocomplexes were recognized on the basis of the fluorescent first-order streaks of the QDs. Model biomarkers in buffer and in 12.6% blank plasma were quantified for validation. The limits of detection for CEA, PSA, and AFP in buffer were in dozens of femtomolar and were close to those in blank plasma. The results demonstrated that our approach worked well in distinguishing immunocomplexes from free and nonspecifically bound probes. The successful quantification of the three targets in five human plasma samples verified the reliability of our method in clinical applications.

Low-Numerical Aperture Microscope Objective Boosted by Liquid-Immersed Dielectric Microspheres for Quantum Dot-Based Digital Immunoassays.

Quantum dot (QD)-based digital immunoassays play an important role in ultrasensitive biomarker detection. However, the requirement of an objective with a high numerical aperture (NA) limits the application of this immunoassay. Here, high-quality imaging of massive single-QDs was achieved by the combination of an air objective (20×/0.4 NA) and liquid-immersed microspheres (150 µm, n = 2.2). The signal-to-noise ratio was comparable to that of a 100×/1.4 NA oil objective. Digital analysis of prostate-specific antigen (PSA) was performed within the dynamic range of 0-50 ng/mL and a limit of detection of 0.17 ng/mL. The measured serum data from the PSA were close to the values provided by a hospital. Using a low-magnification and low-NA objective may reduce the barrier of microscopy miniaturization and is beneficial to popularize biomolecular digital analysis.

Digital Duplex Homogeneous Immunoassay by Counting Immunocomplex Labeled with Quantum Dots.

Digital multiplexed homogeneous immunoassay is supposed to have the advantages of high sensitivity, high analytical throughput, small sampling errors, and low consumption. We present a spectral imaging-based multiplex, homogenous immunoassay by counting sandwich-structured immunocomplexes in the form of quantum dot (QD) aggregates. As a proof of concept, the method was utilized to detect two tumor biomarkers: carcino-embryonic antigen (CEA) and α-fetoprotein (AFP). The immunocomplex induced by CEA contained QD 655 and QD 585 and were recognized by the spectral pattern of dual-color QD aggregates under a transmission-grating-based spectral imaging microscope. Immunocomplexes induced by AFP were labeled with the QD 585 aggregate and were identified by the spectral blue-shift pattern of same-color QD aggregates. Limits of detection for AFP and CEA were calculated to be 0.02 and 0.10 pM at a signal-to-noise ratio of 3, respectively. Further successful quantification of the model proteins in human plasma demonstrated the accuracy and reliability of our approach.

A homogeneous digital biosensor for circulating tumor DNA by the enumeration of a dual-color quantum dot complex.

Monitoring ctDNA in blood is important for cancer management. Here, a one-step single particle counting approach was developed for directly quantifying ctDNA in plasma. Hairpin DNA containing a triple helix stem was immobilized onto QD 585 as a probe. The hairpin was opened by the target, and therefore hybridized with assistant DNA on QD 655, resulting in an aggregate of QD 585 and QD 655. The two-color QD aggregate was regarded as the target. Observed under a single particle transmission grating-based spectral microscope, the two-color QD aggregate was distinguished by a unique spectral pattern of two first-order streaks, and it was counted. The difference in the responses of the probes to perfect-match DNA, single-base mismatch DNA, and non-match DNA indicated that the probe had sufficient single-base discrimination capabilities. The success in plasma recovery tests demonstrated the feasibility of carrying out the direct detection of ctDNA in plasma.

Nonstochastic Protein Counting Analysis for Precision Biomarker Detection: Suppressing Poisson Noise at Ultralow Concentration.

Protein counting analysis obtains the quantitative results of specific protein through counting the number of target signals and displays a great value in disease diagnosis. Current protein counting techniques just stochastically count a small portion of the target signal, which causes a considerable information loss and limits the accuracy and precision of the protein assay at ultralow concentration. Here, we present a nonstochastic and ultrasensitive protein counting method through combining multiround evaporation-induced particle sedimentation, grid-assisted multiframe imaging, and microsphere-enhanced high-resolution signals. Using carcinoembryonic antigen (CEA) as the model, the dynamic range was from 5 × 10-18 M (aM) to 5 × 10-16 M, and the limit of detection was 4.9 aM. For CEA-spiked plasma detection, the relative standard deviation and the relative error of CEA concentrations were both lower than 8.0%, and the recoveries reached 92.5% and 98.8% for 20.0 aM and 40.0 aM CEA respectively. Two clinical plasma samples were measured by the standard addition method, and the results showed little deviation with the values provided by the hospital. The established approach suppresses Poisson noise of the stochastic counting, offers ultrahigh sensitivity, and features a remarkable potential in early disease screening.

Single Gold Nanoparticle-Based Colorimetric Detection of Picomolar Mercury Ion with Dark-Field Microscopy.

Mercury severely damages the environment and human health, particularly when it accumulates in the food chain. Methods for the colorimetric detection of Hg(2+) have increasingly been developed over the past decade because of the progress in nanotechnology. However, the limits of detection (LODs) of these methods are mostly either comparable to or higher than the allowable maximum level (10 nM) in drinking water set by the US Environmental Protection Agency. In this study, we report a single Au nanoparticle (AuNP)-based colorimetric assay for Hg(2+) detection in solution. AuNPs modified with oligonucleotides were fixed on the slide. The fixed AuNPs bound to free AuNPs in the solution in the presence of Hg(2+) because of oligonucleotide hybridization. This process was accompanied by a color change from green to yellow as observed under an optical microscope. The ratio of changed color spots corresponded with Hg(2+) concentration. The LOD was determined as 1.4 pM, which may help guard against mercury accumulation. The proposed approach was applied to environmental samples with recoveries of 98.3 ± 7.7% and 110.0 ± 8.8% for Yuquan River and industrial wastewater, respectively.

Highly sensitive and specific detection of human papillomavirus type 16 using CRISPR/Cas12a assay coupled with an enhanced single nanoparticle dark-field microscopy imaging technique.

Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer. Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques. Ag-Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag-Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods. Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.

Single gold nanoparticle localized surface plasmon resonance spectral imaging for quantifying binding constant of carbohydrate-protein interaction.

Quantifying carbohydrate-protein (ligand-receptor) interactions is important to understand diverse biological processes and to develop new diagnostic and therapeutic methods. We develop an approach to quantitatively study carbohydrate-protein interactions by Au nanoparticle localized surface plasmon resonance (LSPR) peak position shift at the single particles level. Unlike the previous techniques for single particle LSPR spectral imaging, only the first-order streak of an individual nanoparticle is needed to extract a LSPR spectrum, which has great potential to increase throughput to 500 single particle spectra in each frame. LSPR peak shift of protein modified single Au nanoparticles is found to be a function of its ligand concentration, which can be used to fit the binding constants of the interactions. The moderate interactions of Antithrombin III (AT III) and heparins including low molecular weight heparin (LMWH) are determined as well as the strong interaction of transferrin and antitransferrin and the weak interaction of bovine serum album (BSA) and heparin. The measured binding constants of transferrin to antitransferrin, heparin and LMWH to AT III, and BSA to heparin are (3.0 ± 0.6) × 10(9) M(-1), (3.1 ± 0.3) × 10(6) M(-1), (8.0 ± 0.5) × 10(5) M(-1), and (5.1 ± 0.1) × 10(3) M(-1), respectively, which are in good agreement with the reported values.

Photobleaching of quantum dots by non-resonant light.

Core-shell quantum dots suffer from photobleaching by light at wavelengths longer than their emission wavelengths. That is, QD photobleaching can be triggered by photons with low energies that are insufficient to pump electrons into the conduction band. The most probable reason is that electrons are pumped into a surface state and then nonradiatively decayed as in conventional photobleaching.

Corrigendum: Contactless and robust dielectric microspheres-assisted surface-enhanced Raman scattering sensitivity improvement for anthrax biomarker detection.

[This corrects the article DOI: 10.3389/fchem.2022.1057241.].

Superlocalization spectral imaging microscopy of a multicolor quantum dot complex.

The key factor of realizing super-resolution optical microscopy at the single-molecule level is to separately position two adjacent molecules. An opportunity to independently localize target molecules is provided by the intermittency (blinking) in fluorescence of a quantum dot (QD) under the condition that the blinking of each emitter can be recorded and identified. Herein we develop a spectral imaging based color nanoscopy which is capable of determining which QD is blinking in the multicolor QD complex through tracking the first-order spectrum, and thus, the distance at tens of nanometers between two QDs is measured. Three complementary oligonucleotides with lengths of 15, 30, and 45 bp are constructed as calibration rulers. QD585 and QD655 are each linked at one end. The measured average distances are in good agreement with the calculated lengths with a precision of 6 nm, and the intracellular dual-color QDs within a diffraction-limited spot are distinguished.

Contactless and robust dielectric microspheres-assisted surface-enhanced Raman scattering sensitivity improvement for anthrax biomarker detection.

This report presents a contactless and robust dielectric microspheres (DMs)-assisted surface enhanced Raman scattering (SERS) enhancement method to improve SERS detection sensitivity detection sensitivity. DMs that could focus and collect light were embedded within the polydimethylsiloxane (PDMS) film to avoid direct contact with the analytical solution and improve detection reliability. The as prepared DMs embedded PDMS (DMs-PDMS) film was integrated with a microfluidic technique to enhance the SERS signal of a liquid substrate. Detection in microfluidic systems can reduce reagent consumption, shorten assay time, and avoid evaporation of the colloid substrate solution. The robustness and potential influencing factors of DMs-PDMS film assisted SERS enhancement (DERS) were evaluated using 4-aminothiophenol (4-ATP) as the Raman probe. The sensing performance of the proposed method toward dipicolinic acid (DPA) was evaluated, and an evident signal intensification was obtained. Remarkably, the DMs-PDMS film can also be implemented on solid substrates. A proof-of-concept experiment was performed by covering the DMs-PDMS film directly over an AgNPs@Si solid substrate wherein a 5.7-fold sensitivity improvement was achieved.

Aqueous two-phase systems evolved double-layer film for enzymatic activity preservation: A universal protein storage strategy for paper based microdevice.

Integration and storage of bioactive reagents is an important and challenging task in microfluidic paper-based analytical devices (µPADs). Here, we developed a convenient and universal method to store proteins and preserve their activities in µPADs by using aqueous two-phase systems (ATPs) evolved film. A polyethylene glycol (PEG)-dextran (DEX) double-layer film was formed through dehydration of ATPs. Functional biomolecules were stored in the bottom DEX layer on the basis of the biased partitioning and rehydrated conveniently by simple addition of buffer solution at usage. As a demonstration, enzyme immunoassay (EIA) of carcinoembryonic antigen was performed successfully on µPAD integrated with antibodies. Even after 104 days of storage at 4 °C and ambient conditions, the EIA signal just lost less than 10% and 30%, which meet the storage requirements of invitro diagnosis reagents. The ATPs evolved double-layer film has double functions of stabilization and insulation, and provide a high efficiency of biomolecule preservation, thereby promoting the applications of µPADs in POC diagnostic assay.

Optical detection systems on microfluidic chips.

Optical detection continues to dominate detection methods in microfluidics due to its noninvasive nature, easy coupling, rapid response, and high sensitivity. In this review, we summarize two aspects of recent developments in optical detection methods on microfluidic chips. The first aspect is free-space (off-chip) detection on the microchip, in which the conventional absorption, fluorescence, chemiluminescence, surface plasmon resonance, and surface enhanced Raman spectroscopies are involved. The second aspect is the optofluidic (inside-chip) detection. Various miniaturized optical components integrated on the microfluidic chip, such as waveguide, microlens, laser, and detectors are outlined.

Ball-lens assisted sensitivity improvement of fluorescence immunoassay in microchannels.

The relatively low sensitivity is the limitation of ELISA in early screening of diseases. Various signal enhancing methods are developed to increase detection sensitivity, but most of them require professional operation and are not compatible with commercial reagent kits or automatic analysis instruments, limiting their application in clinical testing. Here, we present a contactless and user-friendly ball-lens assisted fluorescence enhancing (BFE) method to improve immunoassay sensitivity. The BFE effect is observed in experiment and demonstrated by the simulation. Based on the BFE effect, the detection sensitivity of the immunoassay is improved about 3.6-fold and the limit of detection is lowered by more than 3-fold using commercial reagents and standard ELISA processes. In addition, the BFE effect causes no damage to the linear dependence, specificity and accuracy of clinical plasma measurement. The established method shows a high compatibility with automatic microfluidic immunoassay systems and other signal enhancing techniques, displaying great potential in multi-technique coupling assays and clinical applications.

Observing photophysical properties of quantum dots in air at the single molecule level: advantages in microarray applications.

Quantum dots (QDs) are promising fluorescent tags for microarrays. Because most microarrays are analyzed under dry conditions, it is necessary to examine the photo properties of QDs in air. We demonstrate that the photophysical characteristics of individual quantum dots are different at the liquid/solid interface compared with QDs at the air/solid interface by observing them through a wide-field fluorescence microscope. QDs in air show higher photo-stability, higher fluorescence signal, slower spectral blue shift rate, less blinking and shorter bulk fluorescence lifetime than those in solution. These beneficial properties indicate QDs are good alternative fluorescent probes for microarrays.

Scattering imaging of single quantum dots with dark-field microscopy.

Scattering images of single quantum dots (QDs) are obtained with a standard dark-field microscope at a video rate. The counts of QDs under dark-field remained constant while the scattering intensity decreased, providing direct proof of quantum dot photophysical bleaching as opposed to desorption or photodecomposition.

Inhibition of photobleaching and blue shift in quantum dots.

Photobleaching and spectral diffusion (blue shift) of quantum dots at the solid/liquid interface are suppressed by adding mercaptoethylamine.