Organic-inorganic hybrid nanoflowers as a new biomimetic platform for ROS-induced apoptosis by photodynamic therapy.

We report here a newly and facile synthesis of the phospholipids@gold nanoflowers (AuNFs) from intact cells as a new biomimetic organic-inorganic hybrid. The most appealing feature of this nanostructure is its dual-absorbing peak in near infrared (NIR) and visible region of spectra, which makes them a potential light-sensitive agent for reactive oxygen species (ROS)-induced apoptosis. Here, in contrast to previous studies, proposed nanostructures are synthesized in a one-pot reaction using phospholipids present in living cell membranes (as a donor cell) with detectable micro process of AuNF formation. The properties of the resulting AuNFs were evaluated through transmission electron microscopy (TEM), as well as FT-IR, 31P-NMR spectra and UV-Vis spectroscopy. Designed cell membrane-based nanostructure looks like an intact cell and would be able to interact with other cells (as a target cell) and also capable to produce cytotoxic singlet oxygen under NIR irradiation. Generated ROS act as a key player in initiation of programmed cell death (apoptosis) and progress of cancer photodynamic therapy (PDT). Cellular experiments on breast cancer MCF-7 cells demonstrated that they may be effective as photodynamic therapy agents.

A turn-off fluorimetric -aptasensor for early detection of apoptosis inside the cells.

To detect cytochrome c (Cyt c) as an important biomarker of apoptosis inside the cells, a simple, label-free, fluorometric detection method has been presented. For this purpose, an aptamer/gold nanocluster probe (Aptamer@AuNCs) was produced which could specifically bind to Cyt c leading to fluorescence quenching of AuNCs. The developed aptasensor showed two linear ranges of 1-80 µM and 100-1000 µM and a detection limit of 0.77 µM and 297.5 µM, respectively. This platform was successfully used to assay Cyt c release inside the apoptotic cells and their cell lysate. Aptamer@AuNC due to its enzyme-like properties could replace antibodies in Cyt c detection by conventional blotting techniques.

Exploitation of N-Gene of SARS-CoV-2 to Develop a New Rapid Assay by ASOs@AuNPs.

A naked-eye (equipment-free), label-free (cost-effective), and RNA extraction-free (to speed up) method for SARS-CoV-2 (as a case study of RNA viruses) detection is developed. Here, the DNA is being used as a template for in situ formation of anisotropic gold nanoparticles (AuNPs) without any chemical modification or DNA labeling. In this study, synthesized AuNPs for the direct detection of N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2 are exploited. To this aim, antisense oligonucleotides (ASOs) with an extra poly guanine tail (G12) were designed. Thus, in the presence of its viral target RNA gene and ASOs@AuNPs-RNA hybridization, there was a red shift in its localized surface plasmon resonance (LSPR), and the intensity of the LSPR peak at 690 nm of throat swab samples was compared to the threshold cycle (Ct) of a reverse-transcriptase real-time polymerase chain reaction (RT-qPCR) (as a gold standard). Results suggested that the plasmonic biosensor can detect a very low amount of SARS-CoV-2 with a detection limit close to RT-qPCR. Simplicity of the new conjugation method with hybridization and annealing without amplification and denaturation steps enabled it to perform in a microfluidic paper-based analytical device.

"Plasmonic Nanomaterials": An emerging avenue in biomedical and biomedical engineering opportunities.

BACKGROUND: Plasmonic nanomaterials asnoble metal-based materials have unique optical characteristic upon exposure to incident light with an appropriate wavelength. Today, generated plasmon by nanoparticles has receivedincreasingattention in nanomedicine; from diagnosis, tissue and tumor imaging to therapeutic and biomedical engineering. AIM OF REVIEW: Due to rapid growing of knowledge in the inorganic nanomaterial field, this paper aims to be a comprehensive and authoritative, critical, and broad interest to the scientific community. Here, we introduce basic physicochemical properties of plasmonic nanoparticles and their applications in biomedical and tissue engineering The first part of each division explain the basic physico-chemical properties of each nanomaterial with a graphical abstract. In the second part, concepts by describing classic examples taken from the biomedical and biomedical engineering literature are illustrated. The selected case studies are intended to give an overview of the different systems and mechanisms utilized in nanomedicine. KEY SCIENTIFIC CONCEPTS OF REVIEW: In this communication, we have tried to introduce the needed concepts of plasmonic nanomaterials and their implication in a particular part of biomedical over the last 20 years. Moreover, in each part with insist on limitations, a perspective is presented which can guide a researcher how they can develop or modify new scaffolds for biomedical engineering.

Building Polyvalent DNA-Functionalized Anisotropic AuNPs using Poly-Guanine-Mediated In-Situ Synthesis for LSPR-Based Assays: Case Study on OncomiR-155.

DNA-functionalized gold nanoparticles (DNA-AuNPs) hold great promise for numerous biomedical applications, especially the building of well-defined nanosystems. Previously reported methods for the preparation of DNA-AuNPs all rely on the use of DNA-bearing free thiol or disulfide groups at their 3'/5' ends. But here we report a novel polyvalent DNA-AuNPs conjugation approach by in-situ fast synthesis of AuNPs at the polyguanine (G12 ) strands. As confirmed by both TEM images and gel electrophoresis analysis, many poly G strand can form an individual anisotropic AuNP and so each AuNP functionalized with a dense layer of DNA, resulting in the formation of polyvalent (p)DNA-AuNPs. The general applicability of this novel approach was further verified in hybridization test and UV-Vis spectroscopy results show that pDNA-AuNPs conjugation is more attractive in biomedical diagnosis and specific sequence detection like microRNA-155 by using an extra-strand poly G with "sticky end" that are complementary to the target sequence.

Colorimetric assay of apoptosis through in-situ biosynthesized gold nanoparticles inside living breast cancer cells.

Abnormalities in apoptosis (More or less than normal rate) is a central factor to detect many human disorders such as many types of cancer and to show therapeutic potential of drugs. During apoptosis, oxidation of reduced gluthation (GSH) to its oxidized form, GSSG, results in a decreased GSH to GSSG ratio. Here, we used altered GSH/GSSG redox state and the release of Cyt c from mitochondria to cytosol as an indicator for apoptosis assay. This study reports a visual method for cell apoptosis assay through in-situ biosynthesized gold nanoparticles (AuNPs) inside livingcells, only after adding a sufficient amount of gold ion. After incubation of apoptotic and non-apoptotic cells with chloroauric acid solution, high level of GSH act as reducing agent in formation of AuNPs using thiol inside living non-apoptotic cells. While in the apoptotic cells, what's happening is based on changes in plasmonic coupling between AuNPs embedded along the oxidized gluthation and Cyt c aggregates in cytosol which causes color changes from red to purple. In this study, we successfully reported cell-based (inside a living cells) and cell free (cell lysis) methods for apoptosis assay of breast cancer cells and we achieved very good results in comparison with a standard apoptosis assay procedure. The linear range for MCF-7 cells detection from 30 to 3 × 105 cells/ml was obtained with a detection limit of 30 cells. In addition, the proposed approach is applicable to detect other apoptotic cells.

Aptamer-based colorimetric determination of early-stage apoptotic cells via the release of cytochrome c from mitochondria and by exploiting silver/platinum alloy nanoclusters as a peroxidase mimic.

An enzyme-free aptameric nanosensor is presented for apoptosis assay. The method exploits the peroxidase-mimicking property of silver/platinum alloy nanoclusters (Ag/Pt NCs) and uses a Cyt c binding ssDNA aptamer. An extra-strand polycytosine (C14) aptamer was designed as a template for synthesis of the Ag/Pt NCs. If cell lysate or purified Cyt c is placed in a polystyrene microplate, Cyt c will bind to the surface of the wells of a microtiterplate. On addition of Apt@Ag/PtNCs, it will associate with Cyt c and then catalytically oxidize colorless tetramethylbenzidine (TMB) in the presence of H2O2 to give a blue colored oxidation product (TMBox) due to the peroxidase-mimicking property of the Ag/Pt NCs. Under optimal conditions, the absorbance of TMB at 660 nm is linearly enhanced as the concentration of Cyt c increases from 50.0 fM to 500 nM, and the detection limit is ~10 pM. The assay is simple, sensitive and cost effective in that it is enzyme-free, antibody-free and label-free. Graphical abstractSchematic diagram of the apoptosis assay on the basis of microplate well-coated mitochondrial cytochrome c releasing by using Aptamer@Ag/Pt NCs.

A New Eye Dual-readout Method for MiRNA Detection based on Dissolution of Gold nanoparticles via LSPR by CdTe QDs Photoinduction.

Breast cancer (BC) is the most frequent cancer that affects one in eight women worldwide. Recent advances in early cancer diagnosis anticipates more efficient treatment and prolong patient survival. MicroRNAs expression profiling plays a key role in diagnosis of cancer such as BC in early stages. For the first time we describe direct injection of hot electrons from plasmonic gold nanoparticles (AuNPs) to adsorbed water molecules with photoinduction of CdTe quantum dots (QDs) with emission wavelength at ~560 nm. As a result of hot electrons exiting from AuNPs with red color, gold cations (holes) are gradually discharged (AuNPs dissolution) leading to a colorless solution. Our group applied this phenomenon to propose a spectral method for miRNA recognition based on different responsive disaggregation and aggregation of CdTe QDs interacted with single strand DNA probes and DNA/RNA heteroduplex respectively resulting in a detection limit of 4.4 pM. This method has been applied also for the determination of miR-155 in the human breast carcinoma MCF-7 cells and normal human embryonic kidney cell line (HEK 293).

A unique FRET approach toward detection of single-base mismatch DNA in BRCA1 gene.

Early detection of mutation carriers in predisposing genes such as BRCA1 plays an important role in disease prevention. This work developed a quantum dots-based (QDs-based) fluorescence resonance energy transfer (FRET) technique for the detection of single-base mismatch DNA in BRCA1 gene. The FRET between QDs as the donor and silver nanocluster (AgNCs) as the acceptor was designed by the strong interaction between CdTe QDs with appropriate size and dsDNA through binding to its major groove. The dsDNA was formed by the hybridization of ssDNA labeled to AgNCs with target DNA, which introduced CdTe QDs into the major grooves to place the AgNCs in close proximity to the QDs. The complementary and single-base mismatch DNA led to obviously different FRET signals. The FRET signal linearly correlated to the concentration of single-base mismatch DNA in the range of 1.5 × 10-10-1.0 × 10-6 mol L-1. The proposed method showed a detection limit of 80 pmol L-1 and the sensitivity comparable to the previously reported assays, indicating promising potential for single nucleotide polymorphisms diagnosis in clinical application.

A colorimetric paper sensor for citrate as biomarker for early stage detection of prostate cancer based on peroxidase-like activity of cysteine-capped gold nanoclusters.

Citrate is currently considered a preferred biomarker for the early stage detection of prostate cancer. In the present work, based on the highly efficient catalytic properties of gold nanoclusters, a novel system for optical determination of citrate was successfully established under optimized conditions. Cysteine-capped gold nanoclusters (Cys-AuNCs) are shown to have an intrinsic peroxidase-mimetic activity. In the presence of H2O2, Cys-AuNCs nanostructures are able to catalyse the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) with high efficiency to produce a blue dye (with an absorbance maximum at 650 nm). Citrate has carboxylic and hydroxyl groups that can bind with free amino and free carboxyl cysteine groups via hydrogen bonds, thus creating a coating on the surface of the gold nanocluster and inhibiting the cluster oxidation activity. Accordingly, a visual, sensitive and simple colorimetric method using Cys-AuNCs as peroxidase mimetic was developed for detecting citrate. A suitable linear relationship for citrate was obtained for the range of 0.5 to 1000 µM. The limit of detection (LOD) of the proposed method was calculated as 0.1 µM and the relative standard deviation (RSD) was obtained to be less than 4.0%. Moreover, the biosensor was used to perform a paper assay on a Y-shaped microfluidic device and make use of the distinctive features of microchannels such as short response time, very low reagent volume required, low fabrication cost etc. A detection limit of 0.4 µM was achieved through the paper test and a good linear range was observed between 1.0 µM-10 mM. The proposed method was further applied to citrate detection in the human urine sample.

Colorimetric and energy transfer based fluorometric turn-on method for determination of microRNA using silver nanoclusters and gold nanoparticles.

The authors describe a dual (colorimetric and fluorometric) detection scheme for microRNA. The method is based on the use of fluorescent DNA-modified silver nanoclusters (DNA-AgNCs) and gold nanoparticles (AuNPs). The DNA-AgNCs were linked to AuNPs via the interaction of ssDNA-AgNC probes with AuNPs. This led to quenching of the fluorescence of the DNA-AgNCs (best measured at excitation/emission peaks of 370/450 nm) and also prevents salt-induced aggregation of the AuNPs. Upon addition of microRNA, the DNA on the AgNCs hybridizes with microRNA. This led to the formation of a DNA-AgNC/miRNA hetero-duplex and increase of the distance to the AuNPs. Quenching was suppressed and fluorescence was restored. The presence of microRNA also affected salt-induced particle aggregation as reflected by a color change from red to purple that is visible with bare eyes. The visual detection limit for microRNA is 0.6 nM, and the fluorometric detection limit is 0.4 pM. Graphical abstract Schematic of the colorimetric and fluorometric assay for microRNA (miRNA). It is based on the use of fluorescent DNA-modified silver nanoclusters (AgNCs) and gold nanoparticles (AuNPs).

Visual detection of miRNA using peroxidase-like catalytic activity of DNA-CuNCs and methylene blue as indicator.

In this study, we developed a colorimetric method which is primarily based on the limited amount of redox in response to the miRNA target analyte binding for the determination of the miRNA, which is used as a biomarker to confirm the clinical diagnosis of cancers such as breast cancer. This method is on the basis of the peroxidase-like property of DNA templated copper nanoclusters (DNA-CuNCs). In this research, it was found that the DNA (poly T) stabilized green emitting CuNCs which exhibited enzyme-like peroxidase activity. After hybridization of miRNA with DNA-CuNCs probe and duplex formation, CuNCs catalyzed the oxidation of the methylene blue (MB) substrate, which can interact easily with DNA/miR-155 heteroduplex. Applying the optimal conditions, the absorbance of MB decreased by increasing the target miRNA-155 with in a dynamic range from 1.0 pM to 10.0 nM down to a detection limit (LOD) of 0.6 pM. The DNA-CuNCs/MB complex was designed to develop a facile, cheap, and fast colorimetric assay for detection of miRNA through MB oxidation by DNA stabilized CuNCs.

A novel BRCA1 gene deletion detection in human breast carcinoma MCF-7 cells through FRET between quantum dots and silver nanoclusters.

BRCA1 (breast cancer 1) genomic deletions are the most important founder mutations in breast cancer patients and can be passed to you from your mother or father. Herein, we report a silver nanoclusters-based (AgNCs-based) fluorescence resonance energy transfer (FRET) method for detection of BRCA1 gene deletion. The method relies on the specific hybridization of DNA-AgNCs fluorescent probe to deleted genes and interaction between double stranded DNA-AgNCs and QD, and the signal amplification through energy transfer from fluorescent AgNCs to QDs during FRET. Such fabricated QDs/DNA-AgNCs interaction might be beneficial for the nanomaterials based biosensing methods Under best possible conditions a linear correlation was established between the fluorescence intensity and the concentration of deletion sequence in the range of 5.0 × 10-13-1.0 × 10-9 M with a detection limit of 1.2 × 10-13 M. Using this method, we could effectively determine gene deletions by using the nonamplified genomic DNAs that were extracted from the MCF-7 as a breast cancer cell line.

An approach toward miRNA detection via different thermo-responsive aggregation/disaggregation of CdTe quantum dots.

MicroRNA-155 regulates the expression of 147 target genes that are involved in cancer pathways, and its expression level has been shown to be up-regulated in breast cancer. Thus, it is necessary to investigate the value of microRNA-155 for early diagnosis and prognosis of breast cancer. Here we present a novel and "light shift" spectral method for the detection of miRNA based on different thermo-responsive aggregation/disaggregation of CdTe quantum dots (CdTe QDs) by using single stranded DNA or a DNA/RNA heteroduplex as a template after heat treatment. In this method upon addition of the DNA/RNA heteroduplex, the CdTe QDs aggregate strongly due to their strong interaction with the double stranded nucleic acid, which results in fluorescence quenching. By applying the melting temperature (T m), the DNA/RNA heteroduplex denatures and two strands are dissociated, which disaggregates the QDs, effectively switching to fluorescence emission of QDs. These processes were investigated with Atomic Force Microscopy (AFM) and fluorescence spectroscopy. The proposed method has been used also for the determination of miR-155 in total RNAs extracted from the human breast carcinoma SK-BR-3 cells and normal human embryonic kidney cell line (HEK 293).

Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters.

Here we describe a label-free detection strategy for large deletion mutation in breast cancer (BC) related gene BRCA1 based on a DNA-silver nanocluster (NC) fluorescence upon recognition-induced hybridization. The specific hybridization of DNA templated silver NCs fluorescent probe to target DNAs can act as effective templates for enhancement of AgNCs fluorescence, which can be used to distinguish the deletion of BRCA1 due to different fluorescence intensities. Under the optimal conditions, the fluorescence intensity of the DNA-AgNCs at emission peaks around 440 nm (upon excitation at 350 nm) increased with the increasing deletion type within a dynamic range from 1.0 × 10-10 to 2.4 × 10-6 M with a detection limit (LOD) of 6.4 × 10-11 M. In this sensing system, the normal type shows no significant fluorescence; on the other hand, the deletion type emits higher fluorescence than normal type. Using this nanobiosensor, we successfully determined mutation using the non-amplified genomic DNAs that were isolated from the BC cell line.

Detection of p53 Gene Mutation (Single-Base Mismatch) Using a Fluorescent Silver Nanoclusters.

P53 mutation was detected through the application of a biosensing approach based on the decrease in the fluorescence of oligonucleotide-templated silver nanoclusters (DNA-AgNCs). To this end specific DNA scaffolds of two various nucleotide fragments were used. One of the scaffolds was enriched with two cytosine sequence fragment (C12). This led to DNA-AgNCs with a fluorescence intensity through chemical reduction, while the other scaffold acted as the probe fragment (5- GTAGATGGCCATGGCGCGGACGCGGGTG-3). This latter scaffold selectively bound to the specific p53 site. Thus, resulting AgNCs demonstrated decreased fluorescence upon binding to single-base mismatching targets, and this behavior was found to be linearly proportional to the concentration of mutated p53 from 5 to 350 nM and the approach was found to be able to detect concentrations as low as 1.3 nM.

A new fluorescence turn-on nanobiosensor for the detection of micro-RNA-21 based on a DNA-gold nanocluster.

In this study, DNA/gold nanoclusters (AuNCs) were used to develop an AuNC-based turn-on fluorescence probe for the analysis of mi-RNA-21, which is a potential screening biomarker for cancer detection. AuNCs on a DNA scaffold were prepared through a one-pot wet-chemical route and evaluated by transmission electron microscopy and dynamic light scattering. Experiments revealed that the fluorescence intensity of the DNA-AuNCs showed a gradual increase with the addition of the target species in a concentration range from 1pM to 10 nM. The method had a detection limit of 0.7 pM and was able to discriminate the target species from mismatched mi-RNAs very efficiently. The method was used for the determination of mi-RNA spiked human plasma samples, and was evaluated as a promising nanobiosensor for application in the selective detection of mi-RNA in various biomedical and clinical tests.

Copper nanocluster-enhanced luminol chemiluminescence for high-selectivity sensing of tryptophan and phenylalanine.

A remarkable method for the highly sensitive detection of phenylalanine and tryptophan based on a chemiluminescence (CL) assay was reported. It was found that fluorescent copper nanoclusters capped with cysteine (Cys-CuNCs) strongly enhance the weak CL signal resulting from the reaction between luminol and H2 O2 . Of the amino acids tested, phenylalanine and tryptophan could enhance the above CL system sensitively. Under optimum conditions, this method was satisfactorily described by a linear calibration curve over a range of 1.0 × 10-6 to 2.7 × 10-5  M for phenylalanine and 1.0 × 10-7 to 3.0 × 10-5  M for tryptophan, respectively. The effect of various parameters such as Cys-CuNC concentration, H2 O2 concentration and pH on the intensity of the CL system were also studied. The main experimental advantage of the proposed method was its selectivity for two amino acids compared with others. To evaluate the applicability of the method to the analysis of a real biological sample it was used to determine tryptophan and phenylalanine in human serum and remarkable results were obtained.

Novel Fluorometric Assay for Detection of Cysteine as a Reducing Agent and Template in Formation of Copper Nanoclusters.

This study was designed to develop a highly selective and sensitive method towards fluorimetric sensing of cysteine (Cys) in water and human serum by using copper nanocluster. The Cys-CuNCs were characterized by scanning electron microscopy (SEM), FTIR, fluorescence and UV-Vis analysis. Spectroscopic evidences showed different intensities that were attributed to the different size of Cys-CuNCs. Enhancement in fluorescence intensity of copper nanoclusters with an increase in concentration of cysteine may enable them to be good candidates in detection systems. Selective recognition of cysteine in aqueous and serum samples was achieved during the formation of various copper nanoclusters (Cys-CuNCs) with different size. Under the optimized conditions, two linear range of the nanobiosensor for cysteine were between the 5 µM to 50 µM with detection limit of 2.4 µM and between 60 µM to 500 µM with detection limit of 55 µM. Fluorescence intensity increased with addition of cysteine concentration from 5 to 50 µM. The proposed low-cost nanobiosensor exhibited high reproducibility and good selectivity. It has been used also for the determination of cysteine in human serum samples with recoveries of 97-103 % and RSDs of 1.8-3.6.

Visual detection of cancer cells by colorimetric aptasensor based on aggregation of gold nanoparticles induced by DNA hybridization.

A simple but highly sensitive colorimetric method was developed to detect cancer cells based on aptamer-cell interaction. Cancer cells were able to capture nucleolin aptamers (AS 1411) through affinity interaction between AS 1411 and nucleolin receptors that are over expressed in cancer cells, The specific binding of AS 1411 to the target cells triggered the removal of aptamers from the solution. Therefore no aptamer remained in the solution to hybridize with complementary ssDNA-AuNP probes as a result the solution color is red. In the absence of target cells or the presence of normal cells, ssDNA-AuNP probes and aptamers were coexisted in solution and the aptamers assembled DNA-AuNPs, produced a purple solution. UV-vis spectrometry demonstrated that this hybridization-based method exhibited selective colorimetric responses to the presence or absence of target cells, which is detectable with naked eye. The linear response for MCF-7 cells in a concentration range from 10 to 10(5) cells was obtained with a detection limit of 10 cells. The proposed method could be extended to detect other cells and showed potential applications in cancer cell detection and early cancer diagnosis.