Label-free Fluorescence Research Articles

Redox-regulated synthesis of fluorescent polydopamine nanoparticles for detection of butyrylcholinesterase activity

Butyrylcholinesterase (BChE) is an enzyme which is relevant to a variety of diseases, and often serve as a common biomarker of health. In this work, a novel fluorescence sensor based on redox-regulated synthesis of polydopamine nanoparticles (PDANPs) has been developed for simple and sensitive sensing BChE activity. A facile and rapid one-step approach for the preparation of fluorescent PDANPs uses potassium permanganate to oxidize dopamine. We demonstrated that the fluorescence intensity of PDANPs is dependent on the dose of potassium permanganate. Butyrylcholinesterase catalyzes the hydrolysis of butyrylthiocholine iodide (BTCh) to produce thiolcholine (TCh) which in a redox reaction with potassium permanganate prevents the formation of fluorescent PDANP. As a result, the activity of BChE can be determined in line with changes in the fluorescence of PDANPs. Based on this finding, a convenient and label-free fluorescence sensor for BChE activity was established via redox-control of the fluorescence intensity of PDANPs. A dynamic response range for BChE is acquired within 0.5 ∼ 200 U/L along with a detection limit of 0.047 U/L. Importantly, the proposed method achieves practical application toward BChE in human sera. Moreover, its satisfying performance for screening of inhibitors was also proved. Hence, the proposed sensor holds great potential for cholinesterase-related biomedical investigation.

Selenocystine-Derived Label-Free Fluorescent Schiff Base Nanocomplex for siRNA Delivery Synergistically Kills Cancer Cells.

Chemo and siRNA synergic treatments for tumors is a promising new therapeutic trend. Selenocystine, a selenium analog of cysteine, has been considered a potential antitumor agent due to its redox perturbing role. In this study, we developed a nanocarrier for siRNA based on a selenocystine analog engineered polyetherimide and achieved traceable siRNA delivery and the synergic killing of tumor cells. Notably, we applied the label-free Schiff base fluorescence mechanism, which enabled us to trace the siRNA delivery and to monitor the selenocystine analogs’ local performance. A novel selenocystine-derived fluorescent Schiff base linker was used to crosslink the polyetherimide, thereby generating a traceable siRNA delivery vehicle with green fluorescence. Moreover, we found that this compound induced tumor cells to undergo senescence. Together with the delivery of a siRNA targeting the anti-apoptotic BCL-xl/w genes in senescent cells, it achieved a synergistic inhibition function by inducing both senescence and apoptosis of tumor cells. Therefore, this study provides insights into the development of label-free probes, prodrugs, and materials towards the synergic strategies for cancer therapy.

Detection of aflatoxin B1 with a new label-free fluorescence aptasensor based on PVP-coated single-walled carbon nanohorns and SYBR Gold.

This paper describes a novel fluorescence label-free aptasensor to detect aflatoxin B1 (AFB1) by utilizing SYBR Gold, aptamer, and single-walled carbon nanohorns (SWCNHs). In the presence of AFB1, the conformation of AFB1-specific aptamer went through and the spatialstructure of this specific aptamer was transformed accordingly. Due to the resistance of the transformed aptamer when adsorbed on the surface of SWCNHs, the protection of the fluorescence of SYBR Gold was accomplished. Consequently, concentrations of AFB1 showed a strong association with fluorescence intensity. The detection limit (LOD) of AFB1 was 1.89ng/mL, while the linear range was 5-200ng/mL and fluorescence intensity satisfactorily correlated (R2 = 0.9919) with the logarithm of AFB1 concentration.

Label-free imaging of non-deparaffinized sections of the human kidney to determine tissue quality and signatures of disease.

Label‐free fluorescence imaging of kidney sections can provide important morphological information, but its utility has not been tested in a histology processing workflow. We tested the feasibility of label‐free imaging of paraffin‐embedded sections without deparaffinization and its potential usefulness in generating actionable data. Kidney tissue specimens were obtained during percutaneous nephrolithotomy or via diagnostic needle biopsy. Unstained non‐deparaffinized sections were imaged using widefield fluorescence microscopy to capture endogenous fluorescence. Some samples were also imaged with confocal microscopy and multiphoton excitation to collect second harmonic generation (SHG) signal to obtain high‐quality autofluorescence images with optical sectioning. To adjudicate the label‐free signal, the samples or corresponding contiguous sections were subsequently deparaffinized and stained with Lillie's allochrome. Label‐free imaging allowed the recognition of various kidney structures and enabled morphological qualification for adequacy. SHG and confocal imaging yielded quantifiable high‐quality images for tissue collagens and revealed specific patterns in glomeruli and various tubules. Disease specimens from patients with diabetic kidney disease and focal segmental glomerulosclerosis showed distinctive signatures compared to specimens from healthy controls with normal kidney function. Quantitative cytometry could also be performed when DAPI is added in situ before imaging. These results show that label‐free imaging of non‐deparaffinized sections provides useful information about tissue quality that could be beneficial to nephropathologists by maximizing the use of scarce kidney tissue. This approach also provides quantifiable features that could inform on the biology of health and disease.

A dual-amplification label-free ratiometric fluorescent sensor for accurate monitoring of telomerase activity based on unique intercalation characteristics of dyes toward different DNA structures

Accurate evaluation and reliable quantitation of telomerase activity in various cancer cells is of significant importance to early cancer diagnosis and therapy. All previously reported ratiometric fluorescence telomerase activity assays inevitably required the expensive labeling or sophisticated probe synthesis, which were laborious and time-consuming. To overcome such restrictions, herein we developed, for the first time, a facile and label-free ratiometric fluorescence sensing strategy for accurately and sensitively detecting telomerase activity based on unique intercalation characteristics of dyes toward double-stranded DNA and G-quadruplex, respectively. By taking advantage of the dual amplification method consisting of catalytic hairpin assembly and exonuclease III assisted cleavage reaction, telomerase activity can be quantitatively detected in MCF-7 cells ranging from 50 to 2000 with a detection limit of 16 MCF-7 cells. Furthermore, our approach could distinguish the relative telomerase activity in cancer cells from that in normal cells, as well as in different cancer cells. It was also confirmed that our strategy displayed good analytical performance for screening the inhibitors for telomerase. More importantly, compared to those labeling and/or single signal-based methods for telomerase activity detection, this assay not only effectively circumvented the expensive labeling and complex probe preparation but also avoided false-positive results, thus exhibiting the appealing features of simplicity, low cost, and high accuracy. Overall, our method might be a promising tool in telomerase activity analysis and telomerase-targeted antitumor drugs screening.

Label-free fluorescent aptasensor for chloramphenicol based on hybridization chain reaction amplification and G-quadruplex/N-methyl mesoporphyrin IX complexation.

The use of the broad-spectrum antibiotic chloramphenicol (CAP) in food is strictly regulated or banned in many countries. Herein, for the sensitive, rapid, and specific detection of CAP in milk, a label-free fluorescence strategy was established based on guanine (G)-quadruplex/N-methyl mesoporphyrin IX (NMM) complex formation and hybridization chain reaction (HCR) amplification. In this system, CAP can specifically bind to an aptamer (Apt) to release an Apt-C sequence from double-stranded DNA (Apt·Apt-C). Apt-C, can further hybridize with a functional hairpin DNA probe to release a primer sequence. The released primer sequence causes HCR and the formation of a nicked double-helix polymer, which contains G-quadruplex DNA. The recognition of G-quadruplex DNA by the NMM fluorochrome results in fluorescence enhancement. Consequently, CAP can be quantitatively detected by measuring the fluorescence intensity at 612 nm. The reliability of the aptasensor method was confirmed by comparison with an enzyme-linked immunosorbent assay. The proposed aptasensor was found to have a limit of detection of 0.8 pg mL−1 for CAP. Moreover, when the aptasensor was applied to the detection of CAP in milk samples, the average recoveries were 99.8–108.3% with relative standard deviations of 4.5–5.2%. Thus, this CAP detection method, which is rapid with high sensitivity and selectivity, has considerable potential for a wide range of food analysis applications.

Orange-emissive N,S-co-doped carbon dots for label-free and sensitive fluorescence assay of vitamin B12

N,S-CDs with orange fluorescent emission were synthesized via a hydrothermal method using o-phenylenediamine and thiourea. A novel fluorometric method for the determination of VB12 based on the IFE was established.

Green synthesis of carbon dots using expired agar for a label-free fluorescence signal-amplified detection of ferric ion utilizing oxalate functionalization

Novel fluorescent (FL) ag-oxCDs are derived from expired agar and oxalate. ag-oxCDs are selective for Fe3+ ions detection (LOD ∼ 75 μM) via FL “turn ON” mechanism. FL nanosensor based on agar biomass with upscale potential is developed herein.

Cationic conjugated polymer-based FRET aptasensor for label-free and ultrasensitive ractopamine detection.

A label-free fluorescence resonance energy transfer (FRET) platform based on cationic conjugated polymers and aptamers for ultrasensitive and specific ractopamine detection was constructed. This method exhibited a wide linear range from 0.05 to 500 μM and a low limit of detection of 47 nM, which make it an attractive assay platform for foodborne doping.

A SYBR Gold-based Label-free in vitro Dicing Assay.

In Arabidopsis, DICER-LIKE PROTEIN 3 (DCL3) cuts the substrate pre-siRNA into a product siRNA duplex, encompassing one 23-nt strand and one 24-nt strand. To monitor the separation of the siRNA duplex with only 1-nt difference, we developed this protocol to evaluate the in vitro dicing activity of DCL3. The method can be applied for measuring the lengths of single-stranded RNA separated through denaturing urea polyacrylamide gel electrophoresis (urea PAGE), which are visualized by a label-free fluorescence SYBR Gold, and quantified in a multi-function imager. This label-free method is easy to conduct, has low cost, and lacks the hazard of the traditional radio-labeled method. This method can also be adapted to the other Dicers and small RNAs.

Label- and enzyme-free plasmon-enhanced single molecule fluorescence detection of HIV DNA fragments based on a catalytic hairpin assembly.

We developed a label- and enzyme-free single molecule fluorescence counting strategy for HIV DNA fragments detection. The nucleic acid biosensor consists of a 5' terminal connected with a triangular gold nanoplate, 3' terminal rich in guanine hairpin probe (HP1) and a hairpin probe HP2 complementary to the partial sequence of HP1. Without the existence of the target DNA, the DNA fragment rich in the guanine region is locked in a hairpin structure and cannot form a G-quadruplex, hence NMM exhibits a low fluorescence signal. When the target DNA exists, the hairpin assembly will trigger a strand displacement amplification reaction that produces a great number of G-quadruplexes, and the fluorescence brightness of NMM will be enhanced. The plasmon resonance effect of the triangular gold nanoplates will further amplify the fluorescence signal. This method can analyze the target DNA with high sensitivity and selectivity, and the detection limit is 0.83 fM. The analysis of the HIV DNA fragments in diluted human serum samples was successfully achieved, and the recovery rate was 92%-104%. Because of its easy operation and low cost, it has broad development potential in biochemical analysis and clinical applications.

Raman and fluorescence imaging of phospholipidosis induced by cationic amphiphilic drugs in endothelial cells

Cationic amphiphilic drugs (CADs) are known from lysosomotropism, drug-induced phospholipidosis (DIPL), activation of autophagy, and decreased cell viability, but the relationship between these events is not clear and little is known about DIPL in the endothelium. In this work, the effects of fluoxetine, amiodarone, clozapine, and risperidone on human microvascular endothelial cells (HMEC-1) were studied using a combined methodology of label-free Raman imaging and fluorescence staining. Raman spectroscopy was applied to characterize biochemical changes in lipid profile and their distribution in the cellular compartments, while fluorescence staining (LysoTracker, LipidTOX, LC3B, and JC-1) was used to analyze lysosome volume expansion, activation of autophagy, lipid accumulation, and mitochondrial membrane depolarization. We demonstrated that fluoxetine, amiodarone, and clozapine, but not risperidone, at non-toxic concentrations induced lipid accumulations in the perinuclear and cytoplasmic regions of endothelial cells. Spectroscopic markers of DIPL included a robust increase in the ratio (lipid/(protein + lipid)), an increase in choline-containing lipid, fatty acids, and the presence of cholesterol esters, while starvation-induced activated autophagy revealed a spectroscopic signature associated with subtle changes in the lipid profile only. Interestingly, lysosomal volume expansion, occurrence of DIPL, and activation of autophagy induced by selected CADs all depended on drug-accumulation in acidic pH of lysosome cellular compartments whereas reduced endothelial viability did not, and was attributed to mitochondrial mechanisms as evidenced by a decreased mitochondrial transmembrane potential. In conclusion, drug-induced phospholipidosis in the endothelium did not reduce endothelial viability per se and can be efficiently assayed by Raman imaging.

Label-free fluorescence aptasensor for ochratoxin A using crystal violet as displacement-type probe

• A label-free fluorescent aptasensor was developed for the detection of OTA. • Crystal violet (CV) as a displacement-type fluorescent probe was discovered for OTA aptasensor. • The rapid and high sensitivity detection of OTA in actual red wine was achieved. • An assay kit is developed base on this method, which meet the maximum permissible specified in European Commission. • The designed assay kit showed broad prospects in portable systems and in-field monitoring. Ochratoxin A (OTA) is a widespread, highly toxic, and harmful mycotoxin. It is commonly found in contaminated crops and foods by a fungal infection. Therefore, to avoid the risk of OTA consumption, rapid and sensitive detection methods are highly required. This report proposes a novel label-free fluorescence aptasensor for the immediate, and highly sensitive detection of OTA in red wine by utilizing crystal violet (CV) as a displacement-type fluorescence probe. CV can interact with OTA aptamer in the absence of its target, thus inducing its folding into an antiparallel G-quadruplex, while CV stacks onto the two external G-tetrads of OTA aptamer engendering a strong fluorescence. However, in the presence of OTA, the CV-aptamer complex will be dissociated, thus decreasing the fluorescence intensity. The experimental data denote that the limit of detection of the developed label-free fluorescence aptasensor achieved 0.56 nmol/L, and the linear range was from 0.56 to 7.8 nmol/L. Moreover, an assay kit was designed based on this method, representing a promising strategy for portable systems and in-field monitoring.

Two-photon excited photoluminescence lifetime imaging studies on individual gelatin-coated gold nanorods

• Intrinsic TPE photoluminescence of different-sized gelatin-coated gold nanorods. • Combined fluorescence spectroscopy and FLIM assays on individual nanorods. • Single gold nanorod TPE photoluminescence overlaps the transverse LSPR band. • Effect of laser excitation power and wavelength on the TPE photoluminescence. The intriguing intrinsic photoluminescence (PL) properties of gold nanorods (AuNRs) have been intensively studied over the last years, not only in the linear but also in the non-linear optics regime, boosting their applicability in various fields of nanobiotechnology. In the present work, we investigate the two-photon excited (TPE) intrinsic PL of different-aspect ratio (AR) synthesized AuNRs, coated with gelatin (g@AuNRs), by performing innovative fluorescence spectroscopy - fluorescence lifetime imaging microscopy (FLIM) combined studies on individual g@AuNRs. In the first step, we employ Finite-difference time-domain (FDTD) simulations to confirm the optical response of AuNRs before and after coating with gelatin. While TPE FLIM assays performed under 800 nm excitation expose some interesting aspect regarding the AR-dependent TPE PL emission generated by individual g@AuNRs, spectroscopic measurements reveal that the TPE PL has a lifetime of around 100 ps and overlaps the corresponding transverse plasmonic band. Finally, we evaluate the effect of laser excitation power and wavelength on the TPE PL, pointing towards the important correlation between these parameters and the TPE PL performance of g@AuNR, as possible contrast agents for label-free TPE fluorescence imaging applications.

Antibody-Free Rapid Detection of SARS-CoV-2 Proteins Using Corona Phase Molecular Recognition to Accelerate Development Time.

To develop better analytical approaches for future global pandemics, it is widely recognized that sensing materials are necessary that enable molecular recognition and sensor assay development on a much faster scale than currently possible. Previously developed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) point-of-care devices are based on the specific molecular recognition using subunit protein antibodies and protein receptors that selectively capture the viral proteins. However, these necessarily involve complex and lengthy development and processing times and are notoriously prone to a loss of biological activity upon sensor immobilization and device interfacing, potentially limiting their use in applications at scale. Here, we report a synthetic strategy for nanoparticle corona interfaces that enables the molecular recognition of SARS-CoV-2 proteins without any antibody and receptor design. Our nanosensor constructs consist of poly(ethylene glycol) (PEG)—phospholipid heteropolymers adsorbed onto near-infrared (nIR) fluorescent single-walled carbon nanotubes (SWCNTs) that recognize the nucleocapsid (N) and spike (S) protein of SARS-CoV-2 using unique three-dimensional (3D) nanosensor interfaces. This results in rapid and label-free nIR fluorescence detection. This antibody-free nanosensor shows up to 50% sensor responses within 5 min of viral protein injections with limit of detection (LOD) values of 48 fM and 350 pM for N and S proteins, respectively. Finally, we demonstrate instrumentation based on a fiber-optic platform that interfaces the advantages of antibody-free molecular recognition and biofluid compatibility in human saliva conditions.

Copper ions assisted fluorescent detection of some dithiocarbamates based on nickel nanocluster with aggregation-induced emission enhancement behavior

Pesticide residue contamination has become an urgent issue since it threatens both the natural environment and public health. In this study, a fluorescent method for detecting dithiocarbamate (DTC) compounds was constructed based on novel nickel nanoclusters (Ni NCs) and copper ions (Cu2+). The water-soluble fluorescent Ni NCs were synthesized for the first time through a one-pot method using glutathione as stabilizer and ascorbic acid as reducing agent. The as-prepared Ni NCs exhibited a maximum fluorescence emission at 445 nm when excited by 380 nm. And they displayed aggregation-induced emission enhancement when ethylene glycol was introduced into the nanocluster aqueous solution. Based on the Ni NCs, a label-free fluorescence quenching sensor was established for sensitive and selective detection of DTC compounds with the assistance of Cu2+. The complex formed by DTC and Cu2+ led to fluorescence quenching of Ni NCs through inner filter effect. The sensing method was successfully applied to two typical DTC compounds, thiram and disulfiram, with good linearity over a wide linear range and a low detection limit. Moreover, the proposed approach was capable of thiram detection in real samples, which confirms the potential of this sensing method as a platform for DTC compound detection.

Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative, label-free fluorescence imaging.

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.

A label-free and enzyme-free fluorescent assay for mercury ions based on T-Hg(II)-T nanoladders and DNA-templated silver nanoclusters/graphene oxide nanocomposites

A label-free and enzyme-free signal-on biosensor for Hg(II) ions was constructed based on T-Hg(II)-T nanoladders and fluorescent DNA-templated silver nanoclusters/graphene oxide nanocomposites (DNA-AgNCs/GO). The T-rich sequences (P1 and P2) were used to capture Hg(II) and form the T-Hg(II)-T nanoladders. C6G5C6 was extended at the 5′ end of P1, used as a template for synthesizing fluorescent DNA-AgNCs. In the presence of mercury ions, the T-Hg(II)-T nanoladders significantly enhanced the emission of AgNCs. GO can attract free P1-C6G5C6 and P2 that were not involved in the formation of T-Hg(II)-T nanoladders, thus employed as the energy acceptor to quench the background fluorescence of AgNCs. The fluorescence enhancement induced by mercury ions can be used for quantitative detection of Hg(II) over the range 0.1–30 nM with a detection limit as low as 7.35 pM. This method was used to detect Hg(II) in the spiked samples of Songhua River water and grass carp, with the recoveries of 97.61%− 103.79% and 96.52%− 105.58% respectively, consistent with the results from atomic fluorescence spectrometry. Obviously, in this assay, enzyme-free signal amplification and signal identification are integrated in the ingenious design of nucleic acid sequences, thus achieving the label-free fluorescence detection of mercury ions.

A label-free fluorescence nanosensor based on nitrogen and phosphorus co-doped carbon quantum dots for ultra-sensitive detection of new coccine in food samples

In this paper, an innovative method for the sensitive detection of new coccine using N, P-doped carbon quantum dots (N,P-CQDs) as fluorescent nanosensor is reported for the first time. The sensing mechanism is based on the fluorescence quenching of N,P-CQDs by new coccine through inner filter effect (IFE). N,P-CQDs were prepared by simple hydrothermal treatment of citric acid, phosphoric acid and ethylenediamine. Under the optimal conditions, the new coccine has two good linear responses in the concentration range of 0.2–100 and 100–200 μM, and the detection limits are as low as 24.8 and 9.4 nM, respectively. Our developed nanosensor has been successfully used for the determination of new coccine in food samples with good precision and high accuracy. This work highlights the economic, rapid, simple, selective and ultra-sensitive for new coccine detection, and opens up a new way for the monitoring of new coccine in actual food samples.

Label-free fluorescence predictions from large-scale correlative light and electron microscopy data

Label-free fluorescence predictions from large-scale correlative light and electron microscopy data