Simultaneous detection of small molecules,proteins and microRNAs using single molecule arrays

Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.

The first example of multiplexed detection of proteins, nucleic acids, and small molecules using single molecule measurement methodology.     

Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

A single-molecule assay for multiple microRNA detection.     

Interplay between intrinsically disordered proteins inside membraneless protein liquid droplets

Intrinsically disordered proteins (IDPs) in cells phase separate to form diverse membraneless organelles, which have condensed liquid droplet-like properties and often contain multiple IDPs. However, how potential interactions between different IDPs affect the dynamic behavior of these protein droplets is largely unknown. Here, we develop a rapid IDP clustering system to generate protein droplets with varied residue compositions and examine diverse interacting IDPs inside droplets. Three different IDP droplets actively recruited other diverse IDPs inside droplets with extremely varied enrichment (inside/outside) degrees (over 100-fold variation) under highly crowded conditions. The recruited IDPs were mostly mobile even inside highly immobile droplets. Among the five tested IDPs, the disordered region of Ddx4 helicase with its unique multiple charged residue blocks was noticeably influenced by droplet mobility. We also discovered that droplets of different IDPs could rapidly fuse to each other. Interestingly, some droplets were heterogeneously fused with segregated subcompartments, and this segregation was enhanced by droplet maturation and was more apparent for specific IDP pairs, in which the polar and charged residue compositions are highly different. The present study not only reports multiple peculiar behaviors of interacting IDP pairs inside droplets but also provides valuable information on generating membraneless organelle models with controllable droplet properties.

Membraneless droplets of intrinsically disordered proteins (IDPs) with varied residue compositions uniquely interact with each other as droplets and clients.     

一种用于核酸高灵敏检测的液滴式数字聚合酶链式反应芯片

为了实现对核酸的高灵敏度检测,构建了一种新型的液滴式数字聚合酶链式反应(dd PCR)芯片.该芯片由产生液滴的聚二甲基硅氧烷(PDMS)模块和储存液滴的玻璃腔室构成.实验结果表明,该芯片可以在25 min内产生2×106个直径为20μm的微液滴(体积4.187 p L).利用该芯片定量检测了表皮生长因子受体(EGFR)基因第19号外显子,在DNA浓度为106~101copies/μL范围内呈现良好的线性关系(R2=0.9998);在浓度为106copies/μL的19号外显子野生型DNA中检测105~100copies/μL的突变型DNA,其检测敏感度可达到0.0001%.该方法在同一芯片上实现了液滴产生、核酸扩增和荧光信号读取的功能,在核酸绝对定量及痕量突变基因的检测中具有潜在应用前景.     

Digital LAMP in a sample self-digitization (SD) chip

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource-limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we accomplished the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings.     

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.     

Comparative quantification of nucleic acids using single-molecule detection and molecular beacons

This paper reports a highly sensitive homogenous method for comparative quantification of nucleic acids based on single-molecule detection (SMD) and molecular beacons (MBs). Two different color MBs were used to perform a separation-free comparative hybridization assay for simultaneous quantification of both target and control strands. A fluorescent burst, emitted from a single hybrid when it passes through a minuscule laser-focused region, is detected with high signal-to-noise ratio (SNR) by using single-molecule fluorescence spectroscopy. Targets are quantified via counting of discrete fluorescent bursts. The high SNR achieved in both detection channels overcame the complications of fluorescent variability usually observed in dual-color ensemble measurements. In comparison with the conventional ensemble methods, this method improved the detection limit by 3 orders of magnitude and reduced the probe consumption by 6 orders of magnitude, facilitating a highly sensitive approach for comparative quantification of nucleic acids and offering great promise for genomic quantification without amplification.     

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

A Janus three-dimensional DNA nanomachine was constructed for the simultaneous and sensitive fluorescent detection and imaging of dual microRNAs in the cancer cells.     

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.     

Visible-light triggered templated ligation on surface using furan-modified PNAs

Oligonucleotide-templated reactions are frequently exploited for target detection in biosensors and for the construction of DNA-based materials and probes in nanotechnology. However, the translation of the specifically used template chemistry from solution to surfaces, with the final aim of achieving highly selective high-throughput systems, has been difficult to reach and therefore, poorly explored. Here, we show the first example of a visible light-triggered templated ligation on a surface, employing furan-modified peptide nucleic acids (PNAs). Tailored photo-oxidation of the pro-reactive furan moiety is ensured by the simultaneous introduction of a weak photosensitizer as well as a nucleophilic moiety in the reacting PNA strand. This allows one to ensure a localized production of singlet oxygen for furan activation, which is not affected by probe dilution or reducing conditions. Simple white light irradiation in combination with target-induced proximity between reactive functionalities upon recognition of a short 22mer DNA or RNA sequence that functions as a template, allows sensitive detection of nucleic acid targets in a 96 well plate format.

Pinpoint production of singlet oxygen was exploited for a self-contained light-triggered activation of a pro-reactive furan moiety, allowing selective and templated surface modification by recognition of short 22mer oligonucleotides.     

One-step hexaplex immunoassays by on-line paper substrate-based electrospray ionization mass spectrometry for combined cancer biomarker screening

Mass spectrometry (MS) is attractive as a multiplexed immunoassay readout benefiting from its high sensitivity, speed and mass resolution. Here, a simple paper-based hexaplex immunoassay with an on-line MS readout was proposed, using functionalized paper as the immune substrates, along with rhodamine-based mass tags assembled on gold nanoparticles prepared as the mass probes (MPs). Simultaneous immune capture and labeling were conducted in one step on paper substrates in 96-well plates with a high throughput within 30 minutes, and the on-line efficient dissociation of the mass tags highly facilitated the hexaplex readout of the immune signals by a newly established on-line paper substrate-based electrospray ionization-MS setup. Six MPs were synthesized for the simultaneous quantification of six important cancer protein markers (cancer antigen 15-3, cancer antigen 19-9, carcinoma embryonic antigen, cancer antigen 125, human epididymis protein 4, and alpha fetoprotein) using only 10 μL serum, presenting satisfactory sensitivity, accuracy and specificity. This platform was further tested in screening for the six biomarkers in serum samples of patients with breast, liver and gastric cancers, showing its high potential for sensitive and specific early cancer diagnosis.

On-line paper substrate based electrospray ionization mass spectrometry for hexaplex immunoassays.     

Ultrasensitive small molecule fluorogenic probe for human heparanase

Heparanase (HPA) is a critical enzyme involved in the remodeling of the extracellular matrix (ECM), and its elevated expression has been linked with diseases such as various types of cancer and inflammation. The detection of heparanase enzymatic activity holds tremendous value in the study of the cellular microenvironment, and search of molecular therapeutics targeting heparanase, however, no structurally defined probes are available for the detection of heparanase activity. Here we present the development of the first ultrasensitive fluorogenic small-molecule probe for heparanase enzymatic activity via tuning the electronic effect of the substrate. The probe exhibits a 756-fold fluorescence turn-on response in the presence of human heparanase, allowing one-step detection of heparanase activity in real-time with a picomolar detection limit. The high sensitivity and robustness of the probe are exemplified in a high-throughput screening assay for heparanase inhibitors.

Heparanase, a critical enzyme involved in the remodeling of the extracellular matrix, activates a disaccharide probe HADP to give a strong fluorescence signal.     

一种用于核酸绝对定量检测的高鲁棒性液滴式数字PCR芯片

为满足液滴式数字聚合酶链式反应(PCR)技术对扩增反应过程中稳定保存液滴以及反应后高效检测的核心需求,构建了一种具有过滤气泡和增强荧光信号功能的液滴式数字聚合酶链式反应芯片.该芯片可在10 min内产生20多万个半径约为21μm的液滴.利用"玻璃天花板"的方式构建了独立于芯片主体材料的液滴收集腔,为液滴提供稳定的保存与反应环境;还构建了过滤结构,可有效过滤混入液相中的空气,提高芯片鲁棒性.同时,在液滴收集腔中引入反射层,增强荧光信号,使单个视野荧光成像时间缩短约40%,提高了检测效率.利用该芯片定量检测EGFR基因第21号外显子,检测信号与DNA浓度在10¹～10⁵copies/μL范围内呈现良好的线性关系(R²=0.998).该方案在载玻片大小的芯片上实现了液滴产生、PCR扩增和荧光信号读取,并具有较高的鲁棒性与检测效率,在核酸检测等方面具有应用潜力.     

Confined space design by nanoparticle self-assembly

Nanoparticle (NP) self-assembly has led to the fabrication of an array of functional nanoscale systems, having diverse architectures and functionalities. In this perspective, we discuss the design and application of NP suprastructures (SPs) characterized by nanoconfined compartments in their self-assembled framework, providing an overview about SP synthetic strategies reported to date and the role of their confined nanocavities in applications in several high-end fields. We also set to give our contribution towards the formation of more advanced nanocompartmentalized SPs able to work in dynamic manners, discussing the opportunities of further advances in NP self-assembly and SP research.

This perspective gives an outlook on the design of interparticle confined nanocavities in self-assembled NP systems and their functional relevance.     

Observation of liquid–liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to elucidate the mechanism of LLPS and the subsequent aggregation process. In this study, we showed that ataxin-3, which is associated with Machado–Joseph disease, exhibits LLPS in an intracellular crowding environment mimicked by biopolymers, and proposed that a single droplet formed in LLPS can be quantified using Raman microscopy in a label-free manner. We succeeded in evaluating the protein concentration and identifying the components present inside and outside a droplet using the O–H stretching band of water as an internal intensity standard. Only water and protein were detected to be present inside droplets with crowding agents remaining outside. The protein concentration in a droplet was dependent on the crowding environment, indicating that the protein concentration and intracellular environment should be considered when investigating LLPS. Raman microscopy has the potential to become a powerful technique for clarifying the chemical nature of LLPS and its relationship with protein aggregation.

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes. We have established a method to quantify a single droplet formed by LLPS using the Raman band of water as an internal standard.     

Porous shape-persistent rylene imine cages with tunable optoelectronic properties and delayed fluorescence

A simultaneous combination of porosity and tunable optoelectronic properties, common in covalent organic frameworks, is rare in shape-persistent organic cages. Yet, organic cages offer important molecular advantages such as solubility and modularity. Herein, we report the synthesis of a series of chiral imine organic cages with three built-in rylene units by means of dynamic imine chemistry and we investigate their textural and optoelectronic properties. Thereby we demonstrate that the synthesized rylene cages can be reversibly reduced at accessible potentials, absorb from UV up to green light, are porous, and preferentially adsorb CO₂ over N₂ and CH₄ with a good selectivity. In addition, we discovered that the cage incorporating three perylene-3,4:9,10-bis(dicarboximide) units displays an efficient delayed fluorescence. Time-correlated single photon counting and transient absorption spectroscopy measurements suggest that the delayed fluorescence is likely a consequence of a reversible intracage charge-separation event. Rylene cages thus offer a promising platform that allows combining the porosity of processable materials and photochemical phenomena useful in diverse applications such as photocatalysis or energy storage.

Chiral rylene imine cages combine porosity and tunable optoelectronic properties. They adsorb CO₂ over N₂ with good selectivity and can show an efficient delayed fluorescence.     

Stochastic DNA Walkers in Droplets for Super‐Multiplexed Bacterial Phenotype Detection

Pathogen detection is growing in importance in the global health arena because of the high morbidity and mortality associated with bacterial blood stream infections. In this work, we present stochastic DNA walkers in droplets (SDwalker‐Drop), a one‐step, rapid, and super‐multiplex method for ultrahigh‐throughput bacterial detection. The SDwalkers, by exploiting cascade signal amplification, endow our analytical platform with fast analysis times and single‐cell analysis ability. The autonomous and multiple‐step walking behavior of the SDwalkers provides a super‐multiplex droplet‐encoding strategy by embedding intensity coded barcodes into a sequence of color‐multiplexed barcodes. We realized a theoretical coding capacity of 8³?1=511 and achieved 20 distinct patterns for bacterial phenotype detection and identification. Moreover, our SDwalker‐Drop platform could be readily integrated with a flow cytometer to afford a general approach for super‐multiplexed, high‐throughput biological assays and screening.     

Quantitative interrogation of protein co-aggregation using multi-color fluorogenic protein aggregation sensors

Co-aggregation of multiple pathogenic proteins is common in neurodegenerative diseases but deconvolution of such biochemical process is challenging. Herein, we developed a dual-color fluorogenic thermal shift assay to simultaneously report on the aggregation of two different proteins and quantitatively study their thermodynamic stability during co-aggregation. Expansion of spectral coverage was first achieved by developing multi-color fluorogenic protein aggregation sensors. Orthogonal detection was enabled by conjugating sensors of minimal fluorescence crosstalk to two different proteins via sortase-tag technology. Using this assay, we quantified shifts in melting temperatures in a heterozygous model protein system, revealing that the thermodynamic stability of wild-type proteins was significantly compromised by the mutant ones but not vice versa. We also examined how small molecule ligands selectively and differentially interfere with such interplay. Finally, we demonstrated these sensors are suited to visualize how different proteins exert influence on each other upon their co-aggregation in live cells.

A little leak will sink a great ship! We prepared a series of multi-color protein aggregation sensors and developed a dual-color thermal shift assay to simultaneously and quantitatively report on protein co-aggregation of two different proteins.     

In vivo monitoring of tissue regeneration using a ratiometric lysosomal AIE probe

Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24–48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.

An AIE-active ratiometric probe for the first time achieved the long-term quantification of lysosomal pH during the medaka larva''s caudal fin regeneration.     

Fluorescent small organic probes for biosensing

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems. With this perspective we highlight sensing mechanisms including Förster resonance energy transfer (FRET), intramolecular charge transfer (ICT), photoinduced electron transfer (PeT), excited state intramolecular proton transfer (ESIPT), aggregation induced emission (AIE) and multiple modality fluorescence approaches including dual/triple sensing mechanisms (DSM or TSM). Throughout the perspective we highlight the remaining challenges and suggest potential directions for development towards improved small-molecule fluorescent probes suitable for biosensing.

Small-molecule based fluorescent probes are increasingly important for the detection and imaging of biological signaling molecules due to their simplicity, high selectivity and sensitivity, whilst being non-invasive, and suitable for real-time analysis of living systems.