Ultrasensitive Ratiometric Fluorescence Bioassay for Accurate Detection of Covid-19-Specific Nucleocapsid Protein in Clinical Serum Samples Using Modified Cleavable Mesoporous SiO2 Satellite-Enriched Carbon Dots.

Due to the presence of various autofluorescent compounds in biological samples like serum and the photobleaching of organic fluorophores, fluorescence sensing has limited practical applicability. This study describes the development of an improved ratiometric fluorescence assay to determine the nucleocapsid protein (N protein), one of the most conserved biomarkers of Covid-19 in spiked and serum samples using highly stable buffer-based near IR-dual emission carbon dots (CDs) encapsulated into the cavities of cleavable silica nanocapsule (SNCs) nanocomposite. The cavities of cleavable silica nanocapsules (SNCs) and the formed core-shell CDs@ SNCs were used as a superior reservoir of fluorescent markers produced by cohydrolyzing tetraethyl orthosilicate and diiminosilane linker, which held hundreds of CDs in silica shell frameworks. The SiO2 nanocomposite was modified with an N protein antibody that specifically paired to the receptor binding region of the Cov-19 spike protein subunit. CDs were taken out of SNCs by NaBH4 reduction, and the released CDs exhibited dual emission at 475 and 675 nm when excited at 400 nm. Ratiometric detection is completed over a binding-induced, concentration-dependent immuno-affinity of the N protein that drives the fluorescence quenching phenomenon between the CDs as fluorophore and the AuNPs as quencher. As the N protein concentration increased, the intensity of the red emission (675 nm) dropped, whereas the intensity of the green emission (475 nm) already remained constant, which is due to sandwich immunoassays of CDs around AuNPs. Using the exceptional fluorescent characteristics of CDs and the high selectivity of nanocomposite functionalized with N-protein antibody, the developed assay efficiently eliminates the autofluorescence background interference of serum samples. The fluorescence ratio (I475/I675) provides a limit of detection of 2 pg mL-1 over a linear range of 0.01 to 5 ng mL-1 and exhibits an amplified sensitivity of 54 times compared to conventional immunoassay using CDs as fluorescent labels. With one-step signal amplification and requiring small sample quantities (only 20 µL), this sensing platform can be effectively used for the accurate detection of N protein, and no cross-reactivity is detected in the presence of different interfering agents.

Development of three-dimensional semi-solid hydrogel matrices for ratiometric fluorescence sensing of Amyloid ß peptide and imaging in SH-SY5 cells: Improvement of point of care diagnosis of Alzheimer's disease biomarker.

Alzheimer's is a neurodegenerative disease with high morbidity and mortality in the elderly, so, detection of its biomarker for definite diagnosis of Alzheimer's in the early stage of disease is a challenge. Amyloid beta peptide (Aß) chosen as an Alzheimer's biomarker. Here, we developed novel, semi-solid, three-dimensional hydrogel matrices for ratiometric fluorescence detection of Aß. This assay's great performance stems from the employment of a hybrid conjugate composed of Rhodamine B (RB), Carbon dots (CDs), and an Aß probe entrapped in Polyvinyl alcohol (PVA), and then detection of fluorescence resonance energy transfer (FRET) that occurs in the presence of AuNP/target-Aß, as a result of hybridization. The RB-CDs' fluorescence (at 582 nm and 675 nm under 430 nm excitation) is quenched in the presence of AuNPs, while the ratio of fluorescence (I582/I675) is increased by the addition of Aß target, and shows a linear relationship in the range of 75 pM-250 nM, with a detection limit of 0.5 pM. Furthermore, the assay possesses strong selectivity for Aß compared to other proteins, and different quantities of a human serum sample successfully analyzed with excellent sensitivity, satisfactory precision, and reliability. Due to distribution of Aß in SH-SY5 human neuroblastoma cells, extending this UV-Vis-NIR full-range responsive CDs bio-probe to imaging of Aß in cells. In both fixed and living SH-SY5 cells, the nanoprobe delivers a clear signal to the Aß target. Because of its high sensitivity, selectivity, biocompatibility and affordability, this nanoprobe is a good option for early Alzheimer's disease diagnosis.

Ratiometric enhanced fluorometric determination and imaging of intracellular microRNA-155 by using carbon dots, gold nanoparticles and rhodamine B for signal amplification.

An ultrasensitive and highly reliable ratiometric assay is described for the determination of microRNA-155. It works at the attomolar concentration level and has high selectivity which warrants its potential application in cancer biomarker tracking. The excellent performance of this method results from (a) the use of a hybrid conjugate prepared from Rhodamine B (RhB), carbon dots (CDs) and probe-microRNA, and (b) from the measurement of fluorescence resonance energy transfer (FRET) that is observed in the AuNP/target-microRNA system as a result of RNA hybridization. The dye RhB (emission peak at 580 nm) serves as an internal reference. The sensitivity of this assay is increased by about 30% because of the broad emissions of CDs (489 nm and 665 nm) through a sequential FRET phenomenon. RhB-CDs were covalently bio-conjugated to probe microRNA. In the presence of AuNPs, the fluorescence of the CDs is quenched, while in the presence of microRNA-155, the ratio of fluorescences at 489 and 665 nm (I489/I665) is enhanced again. A linear relationship exists between the ratio of fluorescence and the concentration of microRNA-155 in the range from 1 aM to 0.1 µM, and the detection limit is 0.3 aM. The assay was applied to quantitative studies of target microRNA-155 in multiple pathways associated with cancer progression in biological fluids include human serum samples and cancer cells. The nanoprobe also deliver clear signal to microRNA target in fixed and lived MDA-MB-231 cells. Graphical abstract A ratiometric FRET sensing method used for microRNA-155 detection at aM concentration level using CDs and AuNPs as donor-acceptor respectively and Rhodamine B as amplification reagent. The application of assay for imaging of microRNA-155 in fixed and live MDA-MB-231 cells is demonstrated.

Simultaneous biosensing of CA125 and CA15-3 tumor markers and imaging of OVCAR-3 and MCF-7 cells lines via bi-color FRET phenomenon using dual blue-green luminescent carbon dots with single excitation wavelength.

Here, we developed a facile route to synthesize two CDs with the maximum emission locates at 326 nm and 443 nm under 230 nm excitation. The absolute photoluminescence quantum yield (QY) is as high as 29% and 52% for CD-1 and CD-2 with strong blue-green fluorescence, respectively. The functionalized CDs (CDs-NH2, CDs-COOH) conjugated with antibodies used for simultaneous detection of two tumor-markers using fluorescence resonance energy transfer (FRET) strategy, when MoS2 nanosheets functionalized with ssDNA related aptamers applied as quencher. The response of sensor enhanced gradually with increasing of targets at range 0.05-8 pg/mL with detection limit (DL) 5.0 fg/mL for CA125, while for CA15-3 concentration range was 1.0-35 pg/mL and DL was 0.5 pg/mL. This strategy revealed a stable analytical performance for the detection of OVCAR-3 and MCF-7 cells lines ranging from 1000 to 10,000 cells/mL with DL 2-3 cells in 10 µL of injected sample. When the CDs-Ab was used in selective imaging of MCF-7 and OVCAR-3 cancer cells, a strong PL emission was observed. After incubation times, both the cells size and shape clearly remained without any change. The results demonstrate that the CDs are a potentially excellent bioimaging component in pathological diagnosis, biomedicine research and management of cancer diseases.

A FRET immunosensor for sensitive detection of CA 15-3 tumor marker in human serum sample and breast cancer cells using antibody functionalized luminescent carbon-dots and AuNPs-dendrimer aptamer as donor-acceptor pair.

We proposed a FRET immunosensing for detection of CA15-3 tumor marker by highly biospecific interactions between CA 15-3 antigen and the corresponding antibody and aptamer. In this sandwich type immunoassay, CA15-3 antibody-functionalized carbon dots and AuNPs labeled PAMAM-Dendrimer/aptamer were used as donor/acceptor, respectively. When CA 15-3 Ag was added to homogenous immunoassay, the strong complex interaction between CA15-3 Ab-CA15-3 Ag- aptamer caused in more coming closer carbon dot and AuNPs and more decreasing fluorescence signal. The decreased fluorescence intensity was linear at three ranges including in concentration range 1.1 µUmL-1 to 16 µU mL-1 with regression of R2 = 0.9879, at the concentration range 16 µU mL-1 to 0.163 mU mL-1 with regression of R2 = 0.9944 and at the concentration range 0.163 mU mL-1 to 5.0 mU mL-1 with regression of R2 = 0.9805. The detection limit of the FRET immunoassay was 0.9 µU mL-1. This assay revealed good sensitivity and specificity with MDA-MB-231 breast cancer cells concentrations from 1000 to 40000 cells/mL with correlation coefficient of 0.9955 and detection limit of 300 cells/mL (3 cells in 10 µL of injected sample). In addition, this FRET immunosensing is applicable in diluted human serum. The recovery values were in the range of 95.86-96.97% for CA 15-3 Ag in spiked serum sample with RSD lower than 7.3%. The proposed immunoassay could be a valid model for establishing other immunoassays for detection of different cancer tumor markers with relevant antigens and antibodies.

An amplified comparative fluorescence resonance energy transfer immunosensing of CA125 tumor marker and ovarian cancer cells using green and economic carbon dots for bio-applications in labeling, imaging and sensing.

CA125, is a marker in the clinical diagnosis of several cancers and currently is the best serum-based tumor marker for ovarian cancer. Here, we developed an ultrasensitive antibody-ssDNA aptamer sandwich-type fluorescence immunosensor for CA125 detection. Based on a novel signal amplification strategy the carbon dots (CDs) functionalized with aptamer (CD-aptamer) used as detection probe and PAMAM-Dendrimers/AuNPs was used for covalent attachment of CA125-antibody and completing the sandwich assay method. By measuring of fluorescence resonance energy transfer (FRET) signals between CDs and AuNPs as nanoquenchers, the fluorescence signal quenched during sandwich complex formed between anti-CA125, CA125 and CDs-Aptamer and decreasing of fluorescence response signal is related to CA125 concentrations. Under optimal conditions, the immunosensor exhibited an extremely low calculated detection limit of 0.5fg/mL with wide linear range 1.0fg/mL to 1.0ng/mL of CA 125. The application of the immunosensor for CA125 detection in serum samples and measuring of ovarian-cancer cells was also investigated. The immunosensor revealed good sensitivity and specificity with ovarian cell concentrations from 2.5×103 to 2×104cells/mL with correlation coefficient of 0.9937 and detection limit of 400cells/mL (4 cell in 10µL), indicating potential application of immunosensor in clinical monitoring of tumor biomarkers. Furthermore, the cell viability was not changed upon treatment with CDs probe during 24h, showing the low cytotoxicity of the probe. More importantly, CDs-antibody hybrid was achieved in selective imaging of the cancer cells over the OVCAR-3 line cells, implying its potential applications in biosensing, as well as in cancer diagnosis.