Quantum Dot Barcodes Research Articles

Genotyping SARS-CoV-2 Variants Using Ratiometric Nucleic Acid Barcode Panels.

Designing diagnostic assays to genotype rapidly mutating viruses remains a challenge despite the overall improvements in nucleic acid detection technologies. RT-PCR and next-generation sequencing are unsuitable for genotyping during outbreaks or in point-of-care detection due to their infrastructure requirements and longer turnaround times. We developed a quantum dot barcode multiplexing system to genotype mutated viruses. We designed multiple quantum dot barcodes to target conserved, wildtype, and mutated regions of SARS-CoV-2. We calculated ratios of the signal output from different barcodes that enabled SARS-CoV-2 detection and identified SARS-CoV-2 variant strains from a sample. We detected different sequence types, including conserved genes, nucleotide deletions, and single nucleotide substitutions. Our system detected SARS-CoV-2 patient specimens with 98% sensitivity and 94% specificity across 91 patient samples. Further, we leveraged our barcoding and ratio system to track the emergence of the N501Y SARS-CoV-2 mutation from December 2020 to May 2021 and demonstrated that the more transmissible N501Y mutation started to dominate infections by April 2021. Our barcoding and signal ratio approach can genotype viruses and track the emergence of viral mutations in a single diagnostic test. This technology can be extended to tracking other viruses. Combined with smartphone detection technologies, this assay can be adapted for point-of-care tracking of viral mutations in real time.

Magnetic quantum dots barcodes using Fe3O4/TiO2 with weak spectral absorption in the visible region for high-sensitivity multiplex detection of tumor markers

Magnetic quantum dot (QD) barcode holds great potential for automatic suspension array and rapid point-of-care detection since it enables simultaneous target encoding, enrichment and separation. However, a serious obstacle to enhancing the encoding capacity of magnetic QD microbeads (MBs) is the fluorescence quenching of magnetic nanoparticles (MNPs) to quantum dots (QDs) in the visible wavelength range due to the broad and strong optical absorption spectrum of MNPs. Here, we report Fe3O4/TiO2 core/shell MNPs and CdSe/ZnS QDs for the construction of dual-function magnetic QD barcodes. Fe3O4/TiO2 MNPs can significantly inhibit fluorescence quenching because the weak absorption of visible light by the TiO2. The two-dimension barcode library of 30 magnetic QD barcodes was constructed based on Fe3O4/TiO2 MNPs and CdSe/ZnS QDs. Moreover, the magnetic QD barcodes showed high sensitivity for the multiplex detection of four tumor markers, cancer antigen 125 (CA125), cancer antigen 199 (CA199), alpha-fetoprotein (AFP), and neuron specific enolase (NSE) with detection limits of 0.89 KU/L, 0.72 KU/L, 0.05 ng/mL, and 0.15 ng/mL, respectively. This bifunctional magnetic QD barcodes are promising for automatic high-sensitivity multiplex bioassay.

Rapid parallel generation of a fluorescently barcoded drop library from a microtiter plate using the plate-interfacing parallel encapsulation (PIPE) chip.

In drop-based microfluidics, an aqueous sample is partitioned into drops using individual pump sources that drive water and oil into a drop-making device. Parallelization of drop-making devices is necessary to achieve high-throughput screening of multiple experimental conditions, especially in time-sensitive studies. Here, we present the plate-interfacing parallel encapsulation (PIPE) chip, a microfluidic chip designed to generate 50 to 90 μm diameter drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard 384-well microtiter plate. The PIPE chip is used to generate two types of optically barcoded drop libraries consisting of two-color fluorescent particle combinations: a library of 24 microbead barcodes and a library of 192 quantum dot barcodes. Barcoded combinations in the drop libraries are rapidly measured within a microfluidic device using fluorescence detection and distinct barcoded populations in the fluorescence drop data are identified using DBSCAN data clustering. Signal analysis reveals that particle size defines the source of dominant noise present in the fluorescence intensity distributions of the barcoded drop populations, arising from Poisson loading for microbeads and shot noise for quantum dots. A barcoded population from a drop library is isolated using fluorescence-activated drop sorting, enabling downstream analysis of drop contents. The PIPE chip can improve multiplexed high-throughput assays by enabling simultaneous encapsulation of barcoded samples stored in a microtiter plate and reducing sample preparation time.

Surveilling and Tracking COVID-19 Patients Using a Portable Quantum Dot Smartphone Device.

The ability to rapidly diagnose, track, and disseminate information for SARS-CoV-2 is critical to minimize its spread. Here, we engineered a portable smartphone-based quantum barcode serological assay device for real-time surveillance of patients infected with SARS-CoV-2. Our device achieved a clinical sensitivity of 90% and specificity of 100% for SARS-CoV-2, as compared to 34% and 100%, respectively, for lateral flow assays in a head-to-head comparison. The lateral flow assay misdiagnosed ∼2 out of 3 SARS-CoV-2 positive patients. Our quantum dot barcode device has ∼3 times greater clinical sensitivity because it is ∼140 times more analytically sensitive than lateral flow assays. Our device can diagnose SARS-CoV-2 at different sampling dates and infectious severity. We developed a databasing app to provide instantaneous results to inform patients, physicians, and public health agencies. This assay and device enable real-time surveillance of SARS-CoV-2 seroprevalence and potential immunity.

Quantum-dot-encapsulated core-shell barcode particles from droplet microfluidics.

The development of robust quantum dot (QD) barcode particles with specific compositions and simple identification is important to meet the demand for high-throughput assays. Here, we present a multiple-inner phase channel capillary microfluidic approach to generate novel QD-encapsulated core-shell barcode particles with distinctive features for multiplexing analysis. By using different QD dispersed polyethylene glycol diacrylate (PEGDA) solutions as the inner phases, the particles were endowed with hydrogel locked QD cores, which could maintain the dispersed status and provide distinctive identification for the particles. The shells of the barcode particles were silica nanoparticle-dispersed ethoxylated trimethylolpropane triacrylate (ETPTA) resin, which could not only improve the stability and biocompatibility of QDs, but also provide functional groups for immobilization of biomolecules due to the assembling of the silica nanoparticles on their surfaces. Due to the advanced emulsification capability of the capillary microfluidic device, double emulsion templates with multiple inner droplet phases and their resultant multicomponent QD-encapsulated core-shell barcode particles could be continually generated. These particles showed remarkable spectral coding capacity in practice, which make them ideal for biomedical applications.

Polymers mediate a one-pot route for functionalized quantum dot barcodes with a large encoding capacity.

With the increasing demands for high-throughput multiplexed bioassays, quantum dot (QD)-encoded microbeads as biocarriers for various bioreactions have attracted considerable attention. However, three key requirements for these biocarriers are still longstanding issues: a stable fluorescence intensity, a large encoding capacity and abundant surface functional groups. Here, a novel one-pot strategy is developed, generating functionalized QD-encoded microspheres with a strong fluorescence intensity and optical stability. With poly(styrene-co-maleic anhydride) (PSMA) molecules as mediators, the encapsulation of QDs and carboxylation of the bead surface are integrated together, greatly improving the preparation efficiency and guaranteeing their potential application in biodetection. Moreover, the mechanism for preparing QD-doped beads is further proposed, which helps to precisely manipulate the preparation process and accurately encode the beads. Through this approach, a single- and dual-color barcode library of QD-encoded microspheres has been successfully established, which demonstrates their great potential in suspension arrays.

Clinical Validation of Quantum Dot Barcode Diagnostic Technology.

There has been a major focus on the clinical translation of emerging technologies for diagnosing patients with infectious diseases, cancer, heart disease, and diabetes. However, most developments still remain at the academic stage where researchers use spiked target molecules to demonstrate the utility of a technology and assess the analytical performance. This approach does not account for the biological complexities and variabilities of human patient samples. As a technology matures and potentially becomes clinically viable, one important intermediate step in the translation process is to conduct a full clinical validation of the technology using a large number of patient samples. Here, we present a full detailed clinical validation of Quantum Dot (QD) barcode technology for diagnosing patients infected with Hepatitis B Virus (HBV). We further demonstrate that the detection of multiple regions of the viral genome using multiplexed QD barcodes improved clinical sensitivity from 54.9-66.7% to 80.4-90.5%, and describe how to use QD barcodes for optimal clinical diagnosis of patients. The use of QDs in biology and medicine was first introduced in 1998 but has not reached clinical care. This study describes our long-term systematic development strategy to advance QD technology to a clinically feasible product for diagnosing patients. Our "blueprint" for translating the QD barcode research concept could be adapted for other nanotechnologies, to efficiently advance diagnostic techniques discovered in the academic laboratory to patient care.

Integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients.

Inorganic nanoparticles are ideal precursors for engineering barcodes for rapidly detecting diseases. Despite advances in the chemical design of these barcodes, they have not advanced to clinical use because they lack sensitivity and are not cost-effective due to requirement of a large read-out system. Here we combined recent advances in quantum dot barcode technology with smartphones and isothermal amplification to engineer a simple and low-cost chip-based wireless multiplex diagnostic device. We characterized the analytical performance of this device and demonstrated that the device is capable of detecting down to 1000 viral genetic copies per milliliter, and this enabled the diagnosis of patients infected with HIV or hepatitis B. More importantly, the barcoding enabled us to detect multiple infectious pathogens simultaneously, in a single test, in less than 1 h. This multiplexing capability of the device enables the diagnosis of infections that are difficult to differentiate clinically due to common symptoms such as a fever or rash. The integration of quantum dot barcoding technology with a smartphone reader provides a capacity for global surveillance of infectious diseases and the potential to accelerate knowledge exchange transfer of emerging or exigent disease threats with healthcare and military organizations in real time.

Self-healing Encapsulation Strategy for Preparing Highly Stable, Functionalized Quantum-Dot Barcodes

Quantum dot (QD) barcodes are becoming an urgent requirement for researchers and clinicians to obtain high-density information in multiplexed suspension (bead-based) assay. However, how to improve the stability of quantum dot barcodes is a longstanding issue. Here, we present a new self-healing encapsulation strategy to generate functionalized uniform quantum dots barcodes with high physical and chemical stability. This efficient and facile strategy could make porous polymer microspheres self-heal to encapsulate QDs via the thermal motion and interaction of the molecular chains. Consequently, the new strategy solved especially the QDs leakage problem and improved the chemical stability under different pH physiological conditions as well as the longtime storage stability. In the meantime, the encoding capacity and the spatial distribution uniformity of quantum dots could be also improved. Furthermore, immunofluorescence assays for alpha fetoprotein (AFP) detections indicated that carboxyl groups on the surface of QD-encoded microspheres could facilitate efficient attachment of biomacromolecules.

Efficient preparation of magnetic quantum dot barcodes.

Quantum dot (QD)-encoded magnetic barcodes were prepared through a highly efficient membrane emulsification-solvent evaporation approach. Our study demonstrates the great potential of these QD-encoded magnetic barcodes for both magnetic separation and multiplex suspension assays.

Fabrication of metal nanoshell quantum-dot barcodes for biomolecular detection

Summary Quantum dot (QD) barcoded microbeads are a promising technology for high-throughput biodetection applications. Here we developed QD barcodes of a novel formulation to improve the bead stability, fluorescence consistency, targeting agents loading, and analytical sensitivity as well as to simplify the conjugation process. This novel formulation contains a mixed-polymer system in preparing the barcodes and a seed-mediated strategy to grow metal nanoshells on the surface of QD barcodes. The newly designed barcodes exhibited enhanced stability and a two-order improvement in analytical sensitivity compared with barcodes without any metal coating. This sensitivity enabled the barcodes to be used for multiplexed biosensing, for example, to differentiate the deadly malaria pathogen strain Plasmodium falciparum from the less lethal Plasmodium vivax specie in a single vial. Such improvements in QD barcodes properties will allow this multiplexed detection platform to emerge from academic development into broad practical applications.

Automating Quantum Dot Barcode Assays Using Microfluidics and Magnetism for the Development of a Point-of-Care Device

The impact of detecting multiple infectious diseases simultaneously at point-of-care with good sensitivity, specificity, and reproducibility would be enormous for containing the spread of diseases in both resource-limited and rich countries. Many barcoding technologies have been introduced for addressing this need as barcodes can be applied to detecting thousands of genetic and protein biomarkers simultaneously. However, the assay process is not automated and is tedious and requires skilled technicians. Barcoding technology is currently limited to use in resource-rich settings. Here we used magnetism and microfluidics technology to automate the multiple steps in a quantum dot barcode assay. The quantum dot-barcoded microbeads are sequentially (a) introduced into the chip, (b) magnetically moved to a stream containing target molecules, (c) moved back to the original stream containing secondary probes, (d) washed, and (e) finally aligned for detection. The assay requires 20 min, has a limit of detection of 1.2 nM, and can detect genetic targets for HIV, hepatitis B, and syphilis. This study provides a simple strategy to automate the entire barcode assay process and moves barcoding technologies one step closer to point-of-care applications.

Predefinable colorimetric quantum-dot barcodes with simple and express identification algorithm

Specific fluorescent profiles were created by loading quantum-dot (Qdot) mixtures in liquid cores of monodispersed polymer microcapsules, which were used as colorimetric barcodes for small object identification. Since the emission intensities of Qdot-loaded liquid cores maintain a linear relation to the Qdot concentrations and the Qdots in the liquid cores with different emission peaks have no obvious interference, the colorimetric barcodes can be predefined by the compositions of Qdot mixtures. The colorimetric barcodes can be identified easily by recording the emission intensities of the encoded microcapsules at respective Qdot emission peaks with a simple and express algorithm, which are suitable to conduct high-throughput multiplexed assays by flow cytometer for biological screening applications.

Highly Efficient Preparation of Multiscaled Quantum Dot Barcodes for Multiplexed Hepatitis B Detection

Both disease diagnosis and therapeutic treatments require real-time information from assays capable of identifying multiple targets. Among various multiplexed biochips, multiplexed suspension assays of quantum dot (QD)-encoded microspheres are highly advantageous. This arises from the excellent fluorescent properties of the QDs incorporated into these microspheres, thus allowing them to serve as "QD barcodes". QD barcodes can be prepared through various approaches. However, the formulation of improved synthetic techniques that may allow more efficient preparation of QD barcodes with better encoding accuracy still remains a challenge. In this report, we describe a combined membrane emulsification-solvent evaporation (MESE) approach for the efficient preparation of QD barcodes. By combining the advantages of the MESE approach in controlling the barcode sizes with accurate encoding, a three-dimensional barcode library that integrates the signals of the forward scattering, fluorescence 1, and fluorescence 4 channels was established via flow cytometry. The five indexes of hepatitis B viruses were chosen as diagnostic targets to examine the feasibility of the QD barcodes in high-throughput diagnosis. On the basis of showing that singleplex detection is feasible, we demonstrate the ability of these QD barcodes to simultaneously and selectively detect a multitude of diverse biomolecular targets.

Fluorescence-profile pre-definable quantum-dot barcodes in liquid-core microcapsules

Microparticles incorporated with quantum-dot (QD) barcodes for multiplexed bioassays attract a great attention due to their potential applications in drug discovery, gene profiling and clinic diagnostics. However, the existing QD barcodes lack a necessary optical stability to ambient fluids or a repeatability of fluorescent profiles. We developed a new QD barcode by loading an aqueous QD mixture as a liquid core into a monodispersed polymer microcapsule by a microfluidic method to avoid those problems. We found that the QDs in the liquid cores were able to maintain their original characteristics, especially the linear relation between photoluminescence intensity and concentration. In addition, we found that the fluorescent profiles of the QD-loaded liquid cores were the same as those of the QD mixtures before being loaded inside the microcapsules. With these two properties, the QD barcodes can be predefined directly by multiplexing the emission peaks and concentrations of the QDs in the liquid cores. Furthermore, the graphical information from fluorescent images of the microcapsules, such as the sizes and numbers of the QD-loaded liquid cores, offers another dimension for barcoding to increase the coding capacity. We also presented a microfluidic method to manufacture the QD-barcoded microcapsules of size ~30 μm. These pre-definable QD barcodes with stable fluorescent profiles can be used as a platform for various high-throughput screening applications in different bioassay buffers.

Data detection algorithms for multiplexed quantum dot encoding

A group of quantum dots can be designed to have a unique spectral emission by varying the size of the quantum dots (wavelength) and number of quantum dots (intensity). This technique has been previously proposed for biological tags and object identification. The potential of this system lies in the ability to have a large number of distinguishable wavelengths and intensity levels. This paper presents a communications system model for MxQDs including the interference between neighbouring QD colours and detector noise. An analytical model of the signal-to-noise ratio of a Charge-Coupled Device (CCD) spectrometer is presented and confirmed with experimental results. We then apply a communications system perspective and propose data detection algorithms that increase the readability of the quantum dots tags. It is demonstrated that multiplexed quantum dot barcodes can be read with 99.7% accuracy using the proposed data detection algorithms in a system with 6 colours and 6 intensity values resulting in 46,655 unique spectral codes.

Facile single step preparation of high-performance quantum dot barcodes

We demonstrate the facile preparation of highly luminescent Cd1−xZnxSe1−ySy quantum dot (QD)-encoded poly(styrene-co-ethylene glycol dimethacrylate-co-methacrylic acid) beads (PSEMBs) in a straightforward and reproducible manner. The monodisperse mesoporous PSEMBs are first swelled in chloroform. Afterwards, the reaction precursors, composed of Cd, Zn, Se and S, are impregnated into the microspheres. Subsequently, the Cd1−xZnxSe1−ySy QDs are synthesized directly within the polymer beads by thermal decomposition. The results show that a wide range of emission wavelengths (490 nm–606 nm) with a narrow full width at half maximum (FWHM) (<40 nm) is obtained by changing the ratios of the precursors. Confocal microscopy images illustrate that highly uniform, bright fluorescent beads are obtained and the QDs have infiltrated into the entire microsphere. The resulting QD barcodes exhibit remarkable stability against solvent induced QD leaching and chemical induced fluorescence quenching. The QD-encoded PSEMBs are found to be photostable in phosphate buffered saline (PBS) (pH 7.4) for at least 3 weeks. Immunoassay performances for human IgG detection indicate that the QD barcodes can be successfully applied to high-throughput multiplexed biomolecular assays.

Microfluidic Generation of Multifunctional Quantum Dot Barcode Particles

We develop a new strategy to prepare quantum dot (QD) barcode particles by polymerizing double-emulsion droplets prepared in capillary microfluidic devices. The resultant barcode particles are composed of stable QD-tagged core particles surrounded by hydrogel shells. These particles exhibit uniform spectral characteristics and excellent coding capability, as confirmed by photoluminescence analyses. By using double-emulsion droplets with two inner droplets of distinct phases as templates, we have also fabricated anisotropic magnetic barcode particles with two separate cores or with a Janus core. These particles enable optical encoding and magnetic separation, thus making them excellent functional barcode particles in biomedical applications.

Rapid Screening of Genetic Biomarkers of Infectious Agents Using Quantum Dot Barcodes

The development of a rapid and sensitive infectious disease diagnostic platform would enable one to select proper treatment and to contain the spread of the disease. Here we examined the feasibility of using quantum dot (QD) barcodes to detect genetic biomarkers of the bloodborne pathogens HIV, malaria, hepatitis B and C, and syphilis. The genetic fragments from these pathogens were detected in less than 10 min at a sample volume of 200 μL and with a detection limit in the femtomol range. A next step for the advancement of QD barcode technology to the clinic will require validation of the technology with human samples to assess for matrix effects, head-to-head comparison with existing detection method, development of techniques to automate the assay and detection process, and simplification of analytical device for the read-out of the barcode signal. Our study provides an important intermediate step in the translation of QD barcode technology for screening infectious disease agents in the developed and developing world.

Integrated parallel microfluidic device for simultaneous preparation of multiplex optical-encoded microbeads with distinct quantum dot barcodes

In this paper we describe a versatile microfluidic strategy for simultaneous preparation of quantum dot-encoded microbeads with controllable barcodes. This method involves the generation of multiple dispersed solutions carrying stepwise concentration gradients of quantum dots by using a pyramidal microfluidic network, the formation of corresponding droplets in parallel flow-focusing droplet generators, and on-chip solidification of these droplets into microbeads. Using a designed microfluidic device, we simultaneously generated five kinds of fluorescence-encoded alginate hydrogel microbeads with a five-level stepwise concentration gradient of monochromatic quantum dot or five controllable ratios of two different-sized quantum dots. This method has the following features: (1) the barcode adapter compartment substitutes the respective preparation of precursor solutions containing the correct concentrations and ratios of different quantum dots for corresponding barcodes. (2) Identical microbeads with distinct quantum dot barcodes can be simultaneously generated in homogeneous conditions. (3) The microfluidic device can be upgraded by increasing or decreasing the inlets and outlets of the pyramidal network and the number of the parallel flow-focusing droplet generators to meet the requirements for the particular barcodes. We expect that this microfluidic route will facilitate the construction and mass-production of robust and reproducible barcode materials.