Simultaneous Detection Of Multiple Biomarkers Research Articles

19 F Barcoding Enables Multiplex Detection of Biomarkers Associated with Organ Injury and Cancer.

Simultaneous detection of multiple biomarkers in complex environments is critical for the in-depth exploration of different biological processes, which is challenging for many current analytical methods due to various limitations. Herein, we report a strategy of 19 F barcoding which takes the advantages of 19 F's high magnetic resonance (MR) sensitivity, prompt signal response to environmental changes, negligible biological background, quantitative signal output, and multiplex capacity. A set of 19 F-barcoded sensors responding to different biomarkers involved in organ injury and cancer are designed, synthesized, and characterized. With these sensors, we accomplish concurrent assessment of different biomarkers in the samples collected from the mice with drug-induced liver/kidney injury or tumor, illustrating the feasibility of this approach for multiplexed detection of different biomarkers in complex environments during various biological processes.

Phasor Analysis of Fluorescence Lifetime Enables Quantitative Multiplexed Molecular Imaging of Three Probes.

The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, cytosol, or nucleus could be accurately distinguished using FLIM and phasor analysis. Next, we successfully resolved and quantified three probes that were all targeted to cell surface biomarkers. Finally, we demonstrated that lifetime-based quantitation accuracy can be improved through intensity matching of various probe-biomarker combinations, which will expand the utility of this technique. Importantly, we reconstructed images for each individual probe, as well as an overlay of all three probes, from a single FLIM image. Our results demonstrate that FLIM and phasor analysis can be leveraged as a powerful tool for simultaneous detection of multiple biomarkers with high sensitivity and accuracy.

(Invited) Machine Learning for DNA/SWCNT Based Molecular Perceptron: Finding Sequences and Training Sensor Arrays

Single wall carbon nanotube (SWCNT) based biosensors provide opportunities for building an ultra-sensitive biosensing system due to their unique optical properties and strong sensitivity to changes in the local environment. Consequently, much effort has been made to develop SWCNT-based sensors. However, the usual method is based on one-to-one recognition which is a difficult way to detect a variety of molecules. In this study, we describe a sensing system, which we call the Molecular Perceptron, that uses an array of weakly-specific sensors combined with a machine learning model. We show how machine learning algorithms, along with choice of feature representation, can be used both for discovery of special resolving DNA sequences and to predict presence and concentration of biomarkers. DNA/SWCNT hybrids were utilized to optically detect biomarker analytes by observing changes in the fluorescence spectra of each SWCNT. Using the experimental data, machine learning models were trained using three different algorithms: Support Vector Machine, Random Forest, and Artificial Neural Network. We demonstrated this platform in gynecologic cancers, often diagnosed at advanced stages, leading to low survival rates. We investigated the detection of protein biomarkers in uterine lavage samples, which are enriched with certain cancer markers compared to blood. We found that the method enables the simultaneous detection of multiple biomarkers in patient samples, with F1-scores of ~0.95 in uterine lavage samples from patients with cancer. This work demonstrates the potential of perception-like systems for the development of multiplexed sensors of disease biomarkers without the need for specific molecular recognition elements. We also demonstrated that machine learning models trained on relatively small DNA sequence data sets can very accurately predict new resolving DNA sequences.

Wearable Eye Patch Biosensor for Noninvasive and Simultaneous Detection of Multiple Biomarkers in Human Tears.

We develop a novel noninvasive wearable biosensor to simultaneously assay multiple key biomarkers in human tears. The sensor is uniquely designed as a convenient-to-use eye patch that can be easily and comfortably fit under eyes. Different sensing regions on the eye patch are modified with specific chromogenic reagents to selectively determine the hydrogen ion (pH), protein, ascorbic acid, and glucose in tears. The assay takes only one drop of tear (∼20 μL) and a 30 s response time. The resulting color signals from each sensing region on the eye patch are captured and analyzed using a smartphone, and a cloud model based on the internet of things concept is recommended, which can extend the application of the eye patch biosensors in point-of-care testing and health monitoring, especially during the pandemic. Tear detection of the human subjects is performed using the proposed method, and the results display good correlation to parallel measurements using assay kits. The results show the great potential and versatility of the proposed eye patch biosensor for noninvasive determination of diverse tear biomarkers, facilitating the widespread application of the tear-based noninvasive wearable biosensor in emerging personal health care and clinical monitoring fields.

Rational Development of Co‐Doped Mesoporous Ceria with High Peroxidase‐Mimicking Activity at Neutral pH for Paper‐Based Colorimetric Detection of Multiple Biomarkers

AbstractPeroxidase‐mimicking nanozymes have been extensively studied, however, their application is limited by the requirement for an acidic pH. Herein, the development of Co‐doped mesoporous cerium oxide (Co‐m‐ceria) is reported, which operates optimally at a near‐neutral pH and exhibits a peroxidase‐like catalytic efficiency that is 600‐times higher than that of pristine m‐ceria. Density functional theory (DFT) calculations for the application of pristine and various metal‐doped m‐ceria in peroxidase‐like reactions under different pH environments are conducted to select Co as the appropriate dopant. The high peroxidase‐like activity of Co‐m‐ceria under neutral conditions and its mesoporous nature enable its application in a one‐pot cascade reaction system, wherein biomarkers of oxidative enzymes can be detected without altering the pH. Five different oxidative enzymes are immobilized in the pores of Co‐m‐ceria at high loadings, followed by incorporation of the enzyme‐containing Co‐m‐ceria in paper microfluidic devices for the convenient and simultaneous detection of multiple biomarkers. The Co‐m‐ceria‐incorporated paper microfluidic device enables the selective and sensitive determination of multiple biomarkers using a smartphone‐acquired image. This study demonstrates the potential of the rational design of nanozymes and their application in paper microfluidic devices, laying the groundwork for future applications of nanozymes in point‐of‐care testing environments.

An Approach to the Simultaneous Detection of Multiple Biomarkers for the Early Diagnosis of Liver Cancer Using Quantum Dot Nanoprobes

Abstract Biomarker-based early diagnosis of liver cancer is of high clinical value for reducing the mortality rate. However, it has been challenging to establish early detection methods with a single biomarker such as alpha-fetoprotein (AFP) because of limited diagnostic sensitivity and specificity. Therefore, developing multiplexed biomarker detection assays is crucially important for early diagnosis. Yet, simultaneous detection methods involving three or more biomarkers have been scarce. Here we suggest employing the serological biomarker panel of glypican-3 (GPC3), dickkopf-1 (DKK1), and AFP for liver cancer detection. We present a rapid simultaneous detection approach for the biomarker panel labeled with three fluorescent quantum dot nanoprobes (emission wavelengths at 565 nm, 605 nm, and 655 nm). As a proof-of-concept, simultaneous fluorescence detection of the biomarker panel was demonstrated using mixed reference samples containing human recombinant GPC3, DKK1, and AFP antigens. Our simultaneous detection approach conferred a linear range of 0.625-2.5 ng•mL-1 for the entire biomarker panel, which merits further clinical validation for the simultaneous and accurate determination of the biomarker panel in human serum samples.

A perception-based nanosensor platform to detect cancer biomarkers.

Conventional molecular recognition elements, such as antibodies, present issues for developing biomolecular assays for use in certain technologies, such as implantable devices. Additionally, antibody development and use, especially for highly multiplexed applications, can be slow and costly. We developed a perception-based platform based on an optical nanosensor array that leverages machine learning algorithms to detect multiple protein biomarkers in biofluids. We demonstrated this platform in gynecologic cancers, often diagnosed at advanced stages, leading to low survival rates. We investigated the detection of protein biomarkers in uterine lavage samples, which are enriched with certain cancer markers compared to blood. We found that the method enables the simultaneous detection of multiple biomarkers in patient samples, with F1-scores of ~0.95 in uterine lavage samples from patients with cancer. This work demonstrates the potential of perception-based systems for the development of multiplexed sensors of disease biomarkers without the need for specific molecular recognition elements.

Multichannel Paper Chip-Based Gas Pressure Bioassay for Simultaneous Detection of Multiple MicroRNAs.

Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.

Graphene Enabled Low‐Noise Surface Chemistry for Multiplexed Sepsis Biomarker Detection in Whole Blood

AbstractAffinity‐based electrochemical (EC) sensors offer a potentially valuable approach for point‐of‐care (POC) diagnostics applications, and for the detection of diseases, such as sepsis, that require simultaneous detection of multiple biomarkers, but their development has been hampered due to biological fouling and EC noise. Here, an EC sensor platform that enables detection of multiple sepsis biomarkers simultaneously by incorporating a nanocomposite coating composed of crosslinked bovine serum albumin containing a network of reduced graphene oxide nanoparticles that prevents biofouling while maintaining electroconductivity is described. Using nanocomposite coated planar gold electrodes, a sensitive procalcitonin (PCT) sensor is constructed and validated in undiluted serum, which produced an excellent correlation with a conventional ELISA (adjusted r2 = 0.95) using clinical samples. A single multiplexed platform containing sensors for three different sepsis biomarkers—PCT, C‐reactive protein, and pathogen‐associated molecular patterns—is also developed, which exhibits specific responses within the clinically significant range without any cross‐reactivity. This platform enables sensitive simultaneous EC detection of multiple analytes in human whole blood, and it can be applied to detect any target analyte with an appropriate antibody pair. Thus, this nanocomposite‐enabled EC sensor platform may offer a potentially valuable tool for development of a wide range of clinical POC diagnostics.

Three-Dimensional Paper-Based Microfluidic Analysis Device for Simultaneous Detection of Multiple Biomarkers with a Smartphone.

Paper-based microfluidic analysis devices (μPADs) have attracted attention as a cost-effective platform for point-of-care testing (POCT), food safety, and environmental monitoring. Recently, three-dimensional (3D)-μPADs have been developed to improve the performance of μPADs. For accurate diagnosis of diseases, however, 3D-μPADs need to be developed to simultaneously detect multiple biomarkers. Here, we report a 3D-μPADs platform for the detection of multiple biomarkers that can be analyzed and diagnosed with a smartphone. The 3D-μPADs were fabricated using a 3D digital light processing printer and consisted of a sample reservoir (300 µL) connected to 24 detection zones (of 4 mm in diameter) through eight microchannels (of 2 mm in width). With the smartphone application, eight different biomarkers related to various diseases were detectable in concentrations ranging from normal to abnormal conditions: glucose (0–20 mmol/L), cholesterol (0–10 mmol/L), albumin (0–7 g/dL), alkaline phosphatase (0–800 U/L), creatinine (0–500 µmol/L), aspartate aminotransferase (0–800 U/L), alanine aminotransferase (0–1000 U/L), and urea nitrogen (0–7.2 mmol/L). These results suggest that 3D-µPADs can be used as a POCT platform for simultaneous detection of multiple biomarkers.

Recent Advancements in Electrochemical Biosensors for Alzheimer's Disease Biomarkers Detection.

BackgroundIt is estimated that the average time between the diagnosis of Alzheimer’s disease (AD) and the patient’s death is 5-9 years. Therefore, both the initial phase of the disease and the preclinical state can be included in the critical period in disease diagnosis. Accordingly, huge progress has recently been observed in biomarker research to identify risk factors for dementia in older people with normal cognitive functions and mild cognitive impairments.MethodsElectrochemical biosensors are excellent analytical tools that are used in the detection of AD biomarkers as they are easy to use, portable, and can do analysis in real time.ResultsThis review presents the analytical techniques currently used to determine AD biomarkers in terms of their advantages and disadvantages; the most important clinical biomarkers of AD and their role in the disease. All recently used biorecognition molecules in electrochemical biosensor development, i.e., receptor protein, antibodies, aptamers and nucleic acids, are summarized for the first time. Novel electrochemical biosensors for AD biomarker detection, as ideal analytical platforms for point-of-care diagnostics, are also reviewed.ConclusionThe article focuses on various strategies of biosensor chemical surface modifications to immobilize biorecognition molecules, enabling specific, quantitative AD biomarker detection in synthetic and clinical samples. In addition, this is the first review that presents innovative single-platform systems for simultaneous detection of multiple biomarkers and other important AD-associated biological species based on electrochemical techniques. The importance of these platforms in disease diagnosis is discussed.

Integrated individually electrochemical array for simultaneously detecting multiple Alzheimer's biomarkers

Simultaneous detection of multiple biomarkers is benefit for reducing the detection cycles, avoiding the false-positive signals, and providing the cross validation, which provide the opportunity to understand the pathogenic mechanisms and achieve precise early diagnosis. Here, we demonstrate the mini-pillar-based individual electrochemical array for simultaneous detection of multiple biomarkers. On such platform, the mini-pillar could confine the microdroplet as individual and open-channel microreactor, which is extremely helpful for reducing reagent consumption and extracting internal information, and the electrodes array embedded in mini-pillar are integrated on one side to achieve multiple and simultaneous electrochemical sensing. The introduction of gold nanodendrites by electrodeposition has greatly enhanced sensitivity via improving probe-binding capacity and response signals. Sensitive and selective detection of multiple Alzheimer's biomarkers including Tau, ApoE4, Amyloid-β and miRNA-101 on such mini-pillar-based biosensor is also achieved. Such biosensor platform with the advantages of high-yield, high sensitivity, low-waste and multiple signals output shows great promise in sensing multiple biomolecules for disease diagnosis and health monitoring.

Multiplexed detection of biomarkers in lateral-flow immunoassays.

Multiplexed detection of biomarkers, i.e., simultaneous detection of multiple biomarkers in a single assay, is a process of great advantages including enhanced diagnostic precision, improved diagnostic efficiency, reduced diagnostic cost, and alleviated pain of patients. A typical lateral-flow immunoassay (LFIA) is a widely used paper-based immunochromatographic test strip designed to detect a target biomarker through two common formats: sandwich assay and competitive assay. In order to obtain qualitative or quantitative results, a probe with unique optical or magnetic properties is usually employed to characterize the concentration of the target biomarker. The typical LFIA is suitable for point-of-care testing due to its simplicity, portability, cost-effectiveness, and rapid detection of a target biomarker. However, detection of a single biomarker in the typical LFIA is not favorable for high throughput analysis. Therefore, multiplexed detection of biomarkers in LFIAs has been extensively studied in recent years for high throughput analysis. To accomplish multiplexed detection of biomarkers in LFIAs, the most frequently used structure is a test strip with multiple test lines (TLs), where each TL can detect a specific biomarker. An alternative structure, i.e., a multi-channel structure with multiple test strips, where each test strip has one TL for detecting a specific biomarker, is employed for multiplexed detection of biomarkers. Sometimes, a single test strip with only one TL containing different receptors, where each detection receptor corresponds to a specific biomarker, is another structure applied for multiplexed detection of biomarkers. This paper reviews three common structures for multiplexed detection of biomarkers in LFIAs, i.e., a test strip with multiple TLs, a multi-channel structure with multiple test strips, and a test strip with a single TL. Based on the three common structures, different signal detection strategies that include colorimetric detection, fluorescence detection, surface-enhanced Raman scattering detection, and magnetic detection, along with performance and perspectives are discussed.

A multiple signal amplification based on PEI and rGO nanocomposite for simultaneous multiple electrochemical immunoassay

A novel electrochemical immunoassay platform for multi-signal amplification has been constructed and applied to the simultaneous detection of three tumor markers (TMs). The platform was successfully developed by polyethyleneimine (PEI)-polydopamine (PDA)/reduced graphene oxide (rGO) nanocomposites. PDA/rGO as the substrates can simultaneously load with detection antibodies (Ab2) and large amounts of PEI, which PEI were further used to immobilize different signal labels. Three TMs of carcinoembryonic antigen (CEA), prostate specific antigen (PSA) and alpha fetoprotein (AFP) were employed as potential factors. Ferrocenecarboxylic acid (Fc), anthraquinone-2-carboxylic acid (Aq) and acetylsalicylic acid (ASA) were employed as distinguishable signal labels. Electrochemical responses of designed sandwich-type immunosensor were successfully investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The multiplexed immunoassay displayed excellent linear response in the range of 0.102 pg/mL–112 ng/mL for CEA and PSA, 0.117 pg/mL–117 ng/mL for AFP. The detection limit of three TMs were 91 fg/mL, 101 fg/mL and 83 fg/mL for CEA, PSA and AFP, respectively. Moreover, this electrochemical immunoassay platform exhibited excellent reproducibility, sensitivity and selectivity for the simultaneous detection of multiple biomarkers.

Label-Free Optical Detection of Multiple Biomarkers in Sweat, Plasma, Urine, and Saliva.

We report a novel label-free quantitative detection of human performance "stress" biomarkers in different body fluids based on optical absorbance of the biomarkers in the ultraviolet (UV) region. Stress biomarker (hormones and neurotransmitters) concentrations in bodily fluids (blood, sweat, urine, saliva) predict the physical and mental state of the individual. The stress biomarkers primarily focused on in this manuscript are cortisol, serotonin, dopamine, norepinephrine, and neuropeptide Y. UV spectroscopy of stress biomarkers performed in the 190-400 nm range has revealed primary and secondary absorption peaks at near-UV wavelengths depending on their molecular structure. UV characterization of individual and multiple biomarkers is reported in various biofluids. A microfluidic/optoelectronic platform for biomarker detection is reported, with a prime focus toward cortisol evaluation. The current limit of detection of cortisol in sweat is ∼200 ng/mL (∼0.5 μM), which is in the normal (healthy) range. Plasma samples containing both serotonin and cortisol resulted in readily detectable absorption peaks at 203 (serotonin) and 247 (cortisol) nm, confirming feasibility of simultaneous detection of multiple biomarkers in biofluid samples. UV spectroscopy performed on various stress biomarkers shows a similar increasing absorption trend with concentration. The detection mechanism is label free, applicable to a variety of biomarker types, and able to detect multiple biomarkers simultaneously in various biofluids. A microfluidic flow cell has been fabricated on a polymer substrate to enable point-of-use/care UV measurement of target biomarkers. The overall sensor combines sample dispensing and fluid transport to the detection location with optical absorption measurements with a UV light emitting diode (LED) and photodiode. The biomarker concentration is indicated as a function of photocurrent generated at the target wavelength.

Simultaneous immunoassays of dual prostate cancer markers using a SERS-based microdroplet channel

The simultaneous detection of multiple biomarkers plays an important role in the accurate diagnosis of cancer. In this study, we developed a novel, surface-enhanced Raman scattering (SERS)-based microfluidic device for the simultaneous detection of free prostate-specific antigen (f-PSA) and total PSA (t-PSA) markers. A fully automatic droplet-based microfluidic platform for the rapid and sensitive detection of f-PSA and t-PSA was designed. Magnetic immunocomplexes were aligned on one side of the channel using a permanent magnet embedded in the microfluidic device, and parent microdroplets containing magnetic immunocomplexes and supernatant solutions were split into two smaller daughter droplets at the Y-shaped junction of the channel. Then, Raman signals of sequential droplets including supernatant solutions were measured for the quantitative analysis of the PSA markers. Two parallel microfluidic channels were designed and fabricated for the simultaneous detection of f-PSA and t-PSA. Our results showed a good linear response for both PSA markers in the range from 0.05 to 100 ng mL−1. The limits of detection were estimated to be below 0.1 ng mL−1 for both the f-PSA and t-PSA. This SERS-based assay in a microfluidic channel was completed in 10 min without any manual incubation and washing steps. Our method is a very promising clinical tool for PSA-based screening test of prostate cancer.

Coupled Fluorometer-Potentiostat System and Metal-Free Monochromatic Luminophores for High-Resolution Wavelength-Resolved Electrochemiluminescent Multiplex Bioassay.

The sensitive simultaneous detection of multiple biomarkers is critical for the early diagnosis of diseases. Electrochemiluminescence (ECL) offers outstanding advantages, e.g., low background, over other optical sensing techniques. However, multiplexed ECL bioassay is hindered not only by the lack of generally available ECL spectrometers but also by the limited number of biocompatible monochromatic ECL luminophores for decades. Herein, we report addressing these issues by re-examination of the recent tabletop spectrofluorometer coupled potentiostat as a high-resolution ECL spectrum acquisition system and using carbon nitrides as monochromatic luminophores. A wavelength-resolved multiplexing ECL biosensor is demonstrated to simultaneously detect CA19-9 and mesothelin, two pancreatic cancer biomarkers, at a single-electrode interface. This work could initiate new opportunities for more general multiplex ECL biosensors with competitive performances.

Synthesis of Monodisperse Plasmonic Magnetic Microbeads and Their Application in Ultrasensitive Detection of Biomolecules.

Plasmon-enhanced fluorescence (PEF)-based analytical technology has recently demonstrated its ability in detecting biomarkers with ultrahigh sensitivity. However, the scope of the PEF-based technology has been hindered by its reliance on flat substrates with relatively low binding kinetics and the limited multiplex detection ability. Herein, we reported a simple yet robust method for the fabrication of plasmonic magnetic microbeads (PMMBs)-based suspension array technology (SAT) with fluorescence enhancement of about 60-fold, improving the detection limit of biomarkers by 2-orders of magnitude toward 100 fM. We also demonstrated the performance of this method for the detection of anti-acidic ribosomal phosphoprotein 0 (anti-P0) autoantibody in sera from systemic lupus erythematosus (SLE) patients. Owing to the high sensitivity and efficient magnet-based sample collection, our method can be employed for detection of ultrasmall volumes of samples (e.g., 2 μL), promising for point-of-care detection. Furthermore, a size-encoded PMMBs-based multiplexed suspension array for simultaneous detection of multiple biomarkers is realized, illustrating the great potential of this technology in high-throughput disease diagnosis applications.

Peptide-Mediated Controllable Cross-Linking of Gold Nanoparticles for Immunoassays with Tunable Detection Range.

The colorimetric immunoassay based on gold nanoparticles (AuNPs) can hardly enable simultaneous detection of multiple biomarkers in vastly different concentrations (e.g., pg/mL-μg/mL) because of its narrow dynamic range. In this work, we demonstrate an immunoassay with tunable detection range by using peptide-mediated controlled aggregation of surface modification-free AuNPs. Alkaline phosphatase (ALP) removes the phosphate group of the peptide to yield a positively charged product, which triggers the aggregation of negatively charged AuNPs and the color change of the AuNPs solution from red to blue with naked-eye readout. We design and screen 20 kinds of phosphorylated peptides to obtain a broad and controllable detection range for ALP sensing and apply them for detecting multiple inflammatory biomarkers in clinical samples. Our assay realizes straightforward, multiplexed, and simultaneous detection of multiple clinical biomarkers with tunable detection range (from pg/mL to μg/mL) in the same run and holds great potential for chemical/biochemical analysis.

Two-Color-Based Nanoflares for Multiplexed MicroRNAs Imaging in Live Cells.

MicroRNAs (miRNAs) have become an ideal biomarker candidate for early diagnosis of diseases. But various diseases involve changes in the expression of different miRNAs. Therefore, multiplexed assay of miRNAs in live cells can provide critical information for our better understanding of their roles in cells and further validating of their function in clinical diagnoses. Simultaneous detection of multiple biomarkers could effectively improve the accuracy of early cancer diagnosis. Here, we develop the two-color-based nanoflares for simultaneously detecting two distinct miRNA targets inside live cells. The nanoflares consist of gold nanoparticles (AuNPs) functionalized with a dense shell of recognition sequences hybridized to two short fluorophore-labeled DNA molecules, termed “flares”. In this conformation, the close proximity of the fluorophore to the AuNPs surface leads to quenching of the fluorescence. However, when target miRNAs bind to the recognition sequence, the concomitant displacement of the flare can be detected as a corresponding increase in fluorescence. The results demonstrate that the two-color-based nanoflares can simultaneously detect miR-21 and miR-141 expression levels in various live cancer cells successfully. Compared to the traditional single-color-based nanoflares, the two-color-based nanoflares could offer more reliable and practical information for cancer detection, improving the accuracy of early disease diagnosis.