Methylene Blue-Stained Single-Stranded DNA Aptamers as a Highly Efficient Electronic Switch for Quasi-Reagentless Exosomes Detection: An Old Dog with New Tricks.

Improving the convenience, sensitivity, and cost-effectiveness of electrochemical biosensors is crucial for advancing their clinical diagnostic applications. Herein, we presented an elegant approach to construct electrochemical aptasensors for tumor-derived exosome detection by harnessing the alterable interaction between methylene blue (MB) and DNA aptamer. In detail, the anti-EpCAM aptamer, named SYL3C, was found to exhibit a strong affinity toward MB due to the specific interaction between MB and unbound guanine bases. Thereby, SYL3C could be stained with MB to arouse a strong electrochemical signal on a gold electrode (AuE). Upon binding to EpCAM-positive exosomes, SYL3C underwent a conformational transformation. The resulting conformation, or exosomes-SYL3C complex, not only reduced the accumulation of MB on SYL3C by obstructing the accessibility of guanines to MB but also impeded the transfer of electrons from the bound MB to AuE, leading to a notable decrease in the electrochemical signal. Using MB-stained SYL3C as an electronic switch, an electrochemical aptasensor was readily established for the detection of EpCAM-positive exosome detection. Without the need for signal amplification strategies, expensive auxiliary reagents, and complex operation, this unique signal transduction mechanism alone could endow the aptasensor with ultrahigh sensitivity. A limit of detection (LOD) of 234 particles mL-1 was achieved, surpassing the performance of most reported methods. As a proof of concept, the aptasensor was applied to analyze clinical serum samples and effectively distinguish non-small-cell lung cancer (NSCLC) patients from healthy individuals. As EpCAM exhibits broad expression in exosomes derived from different tumor sources, the developed aptasensor holds promise for diagnosing other tumor types.

CaZnO-based nanoghosts for the detection of ssDNA, pCRISPR and recombinant SARS-CoV-2 spike antigen and targeted delivery of doxorubicin.

Overexpression of proteins/antigens and other gene-related sequences in the bodies could lead to significant mutations and refractory diseases. Detection and identification of assorted trace concentrations of such proteins/antigens and/or gene-related sequences remain challenging, affecting different pathogens and making viruses stronger. Correspondingly, coronavirus (SARS-CoV-2) mutations/alterations and spread could lead to overexpression of ssDNA and the related antigens in the population and brisk activity in gene-editing technologies in the treatment/detection may lead to the presence of pCRISPR in the blood. Therefore, the detection and evaluation of their trace concentrations are of critical importance. CaZnO-based nanoghosts (NGs) were synthesized with the assistance of a high-gravity technique at a 1,800 MHz field, capitalizing on the use of Rosmarinus officinalis leaf extract as the templating agent. A complete chemical, physical and biological investigation revealed that the synthesized NGs presented similar morphological features to the mesenchymal stem cells (MSCs), resulting in excellent biocompatibility, interaction with ssDNA- and/or pCRISPR-surface, through various chemical and physical mechanisms. This comprise the unprecedented synthesis of a fully inorganic nanostructure with behavior that is similar to MSCs. Furthermore, the endowed exceptional ability of inorganic NGs for detective sensing/folding of ssDNA and pCRISPR and recombinant SARS-CoV-2 spike antigen (RSCSA), along with in-situ hydrogen peroxide detection on the HEK-293 and HeLa cell lines, was discerned. On average, they displayed a high drug loading capacity of 55%, and the acceptable internalizations inside the HT-29 cell lines affirmed the anticipated MSCs-like behavior of these inorganic-NGs.

Immunofluorescence microscopy-based detection of ssDNA foci by BrdU in mammalian cells.

DNA end resection converts broken ends of double-stranded DNA (dsDNA) to 3'-single-stranded DNA (3'-ssDNA). The extent of resection regulates DNA double-strand break (DSB) repair pathway choice and thereby genomic stability. Here, we characterize an optimized immunofluorescence (IF) microscopy-based protocol for measuring ssDNA in mammalian cells by labeling genomic DNA with 5-bromo-2'-deoxyuridine (BrdU). BrdU foci can be detected under non-denaturing conditions by anti-BrdU antibody, providing an accurate and reliable readout of DNA end resection in most mammalian cell lines. For complete details on the use and execution of this protocol, please refer to Kilgas et al. (2021).

Detection and Separation of Single-Stranded DNA Fragments Using Solid-State Nanopores.

Many biological assays require effectively and sensitively sorting DNA fragments. Here, we demonstrate a solid-state nanopore platform for label-free detection and separation of short single-stranded DNA (ssDNA) fragments (<100 nt), based on their length-dependent translocation behaviors. Our experimental data show that each sized pore has a passable length threshold. The negative charged ssDNA fragments with length smaller than the threshold can be electrically facilitated driven through the correspondingly sized nanopore along the direction of electric field. In addition, the passable length threshold increases with the pore size enlarging. As a result, this phenomenon is able to be applicable for the controllable selectivity of ssDNA by tuning nanopore size, and the selectivity limitation is up to 30nt. Numerical simulation results indicate the translocation direction of ssDNA is governed by the competition of electroosmosis and electrophoresis effects on the ssDNA and offer the relationship between passable length threshold and pore size.

McH-lpr/lpr-RA1 mice: A novel spontaneous mouse model of autoimmune sialadenitis.

Many studies of the autoimmune disease Sjögren's syndrome have been performed using spontaneous mouse models. In the present study, we describe the characteristics of McH/lpr-RA1 mice and propose their use as a novel murine model of autoimmune sialadenitis. The McH/lpr-RA1 mouse is a recombinant congenic strain derived from generation F54 or more of MRL-Faslpr x (MRL- Faslpr x C3H- Faslpr) F1. We show for the first time that this mouse spontaneously develops autoimmune sialadenitis and vasculitis in submandibular gland tissues. Sialadenitis was accompanied by extensive inflammatory cell infiltration and tissue destruction. Immunohistochemical studies revealed that the salivary gland lesions strongly expressed four sialadenitis-related molecules: SSA and SSB (autoantigens of Sjögren's syndrome), gp91phox (an accelerator of reactive oxygen species production) and single strand DNA (a marker of apoptotic cells). In contrast, expression of aquaporin-5 (AQP5), which stimulates salivary secretion was weak or negligible. Statistical correlation analyses indicated that the apoptosis of salivary gland cells provoked by oxidative stress contributed to the severe sialadenitis and reduced expression of AQP5. Our study has demonstrated that McH/lpr-RA1 mice spontaneously develop the pathognomonic features of autoimmune sialadenitis and thus could be used as a new animal model of Sjögren's syndrome.

Improving the detection limit of Salmonella colorimetry using long ssDNA of asymmetric-PCR and non-functionalized AuNPs.

A colorimetric sensor based on gold nanoparticles (AuNPs) and single-stranded DNA (ssDNA) is a simple and rapid method for detecting foodborne pathogens. However, the colorimetric method employed in previous studies involved short ssDNA (<100 nucleotides), including the aptamer and PCR products, resulting in the high detection limit of this technique. In this study, a colorimetric sensor was developed based on long ssDNA of asymmetric PCR (aPCR) and non-functionalized AuNPs for detecting Salmonella Typhimurium (S. Typhimurium). In the presence of S. Typhimurium, the long ssDNA (547 nt) amplified by aPCR-protected AuNPs from NaCl-induced aggregation, while the solution retained a red color. After optimizing parameters, the limit of detection (LOD) of the colorimetric sensor was 2.56 CFU/mL with high specificity. Recovery studies showed its feasibility for detecting S. Typhimurium (102 CFU/mL, 104 CFU/mL, and 106 CFU/mL) in spiked lettuce samples. This colorimetric sensor provides new opportunities for the highly sensitive detection of bacteria in real food samples.

Biotechnological production of ssDNA with DNA-hydrolyzing deoxyribozymes.

Preparation of long single-stranded (ss)DNA in large quantities with high efficiency and purity remains a synthetic challenge. Here, we present a protocol for using DNA-hydrolyzing DNA enzymes (deoxyribozymes) for efficient biotechnological production of milligrams of ssDNA with a customizable sequence up to a few kilobases. Our protocol provides a convenient yet economical way to store the sequence information of target ssDNA on phages for selective mass production on demand. For complete details on the use and execution of this protocol, please refer to Jia et al. (2021).

New DNA-hydrolyzing DNAs isolated from an ssDNA library carrying a terminal hybridization stem.

DNA-hydrolyzing DNAs represent an attractive type of DNA-processing catalysts distinctive from the protein-based restriction enzymes. The innate DNA property has enabled them to readily join DNA-based manipulations to promote the development of DNA biotechnology. A major in vitro selection strategy to identify these DNA catalysts relies tightly on the isolation of linear DNAs processed from a circular single-stranded (ss) DNA sequence library by self-hydrolysis. Herein, we report that by programming a terminal hybridization stem in the library, other than the previously reported classes (I & II) of deoxyribozymes, two new classes (III & IV) were identified with the old selection strategy to site-specifically hydrolyze DNA in the presence of Zn2+. Their representatives own a catalytic core consisting of ∼20 conserved nucleotides and a half-life of ∼15 min at neutral pH. In a bimolecular construct, class III exhibits unique broad generality on the enzyme strand, which can be potentially harnessed to engineer DNA-responsive DNA hydrolyzers for detection of any target ssDNA sequence. Besides the new findings, this work should also provide an improved approach to select for DNA-hydrolyzing deoxyribozymes that use various molecules and ions as cofactors.

Testing a series of modifications on genomic library preparation methods for ancient or degraded DNA.

Technological advancements have revolutionized ancient and degraded DNA analysis, moving the field to the Next Generation Sequencing era. One of the advancements, the ancient DNA-oriented high-throughput library preparation methods, enabled the sequencing of more endogenous molecules. Although fairly optimized, both single- and double-stranded library preparation methods hold the potential for further improvement. Here, we test a series of modifications made at different steps of both single- and double-stranded library preparation methods. Given all the modifications tested, we found that two of them provide further benefits, including the use of Endonuclease VIII as a pre-treatment step before preparing single-stranded libraries and the use of a modified second adapter of the single stranded-libraries as an alternative option to enable sequencing of single stranded-libraries with the standard Illumina sequencing primer instead of the custom designed as described in the single stranded library preparation method. Furthermore, we propose uracil-DNA-glycosylase (UDG) could also be considered for both single- and double-stranded library preparation methods, although additional parameters should be taken into account depending on the sequencing strategy and the sample characteristics. Further modifications were also tested and although they were not advantageous, they could be considered as equivalent to the published options.

PPV and Detection Rate of mt-sDNA Testing, FIT, and CT Colonography for Advanced Neoplasia: A Hierarchic Bayesian Meta-Analysis of the Noninvasive Colorectal Screening Tests.

BACKGROUND. Noninvasive tests for colorectal cancer (CRC) screening and prevention limit the need for invasive colonoscopy to follow up positive test results. However, the relative performance characteristics of available noninvasive tests have not yet been adequately compared. OBJECTIVE. We performed a systematic review and meta-analysis to compare the diagnostic performance of the available noninvasive CRC screening tests, including multitarget stool DNA (mt-sDNA) testing, fecal immunochemical testing (FIT), and CT colonography (CTC), with an emphasis on comparison of PPV and detection rate (DR) for advanced neoplasia (AN; encompassing cases of advanced adenomas and CRC). EVIDENCE ACQUISITION. After systematic searches of MEDLINE and Google Scholar databases, 10 mt-sDNA, 27 CTC, and 88 FIT published screening studies involving 25,132, 33,493, and 2,355,958 asymptomatic adults, respectively, were included. Meta-analysis with hierarchic Bayesian modeling was conducted in accordance with Cochrane Collaboration and PRISMA guidelines to determine test positivity rates (TPRs) leading to optical colonoscopy, as well as PPVs and DRs for both AN and CRC. Different positivity thresholds were considered for FIT and CTC. EVIDENCE SYNTHESIS. Point estimates (with 95% credible intervals) from pooled Bayesian meta-analysis combining all thresholds for FIT and stratifying CTC results by a polyp size threshold of 6 mm or larger (CTC6) and 10 mm or larger (CTC10) were calculated. TPR was 13.5% (10.9-16.6%) for mt-sDNA testing, 6.4% (5.8-7.2%) for FIT, 13.4% (11.4-15.6%) for CTC6, and 6.6% (5.2-7.7%) for CTC10. AN PPV was 26.9% (95% credible interval, 21.8-33.2%) for mt-sDNA testing, 31.8% (29.3-34.5%) for FIT, 34.4% (27.2-41.0%) for CTC6, and 61.0% (54.0-70.0%) for CTC10. CRC PPV was 2.4% (1.5-3.9%) for mt-sDNA testing, 4.9% (4.3-5.3%) for FIT, 3.5% (2.5-4.8%) for CTC6, and 6.0% (4.3-8.0%) for CTC10. The DR for AN was 3.4% (95% credible interval, 2.5-4.8%) for mt-SDNA, 2.0% (1.8-2.3%) for FIT, 4.8% (4.0-6.5%) for CTC6, and 4.0% (3.0-4.6%) for CTC10. When FIT is restricted to a lower threshold (< 10 µg Hb/g feces), its performance profile is similar to that of mt-sDNA testing, although available data are limited. AN PPV odds ratios (relative to CTC10 as the reference) were 0.24 (95% credible interval, 0.17-0.33) for mt-sDNA testing, 0.30 (0.24-0.45) for FIT, and 0.33 (0.25-0.47) for CTC6. CONCLUSION. Among noninvasive CRC screening tests, CTC with a polyp size threshold of 10 mm or larger most effectively targets AN, preserving detection while also decreasing unnecessary colonoscopies compared with mt-sDNA testing and FIT. CLINICAL IMPACT. CTC performed with a polyp size threshold for colonoscopy referral set at 10 mm or larger represents the most effective and efficient noninvasive screening test for CRC prevention and detection.

Microheater-integrated zinc oxide nanowire microfluidic device for hybridization-based detection of target single-stranded DNA.

Detection of cell-free DNA (cfDNA) has an impact on DNA analysis in liquid biopsies. However, current strategies to detect cfDNA have limitations that should be overcome, such as having low sensitivity and requiring much time and a specialized instrument. Thus, non-invasive and rapid detection tools are needed for disease prevention and early-stage treatment. Here we developed a device having a microheater integrated with zinc oxide nanowires (microheater-ZnO-NWs) to detect target single-stranded DNAs (ssDNAs) based on DNA probe hybridization. We confirmed experimentally that our device realizedin-situannealed DNA probes by which we subsequently detected target ssDNAs. We envision that this device can be utilized for fundamental studies related to nanobiodevice-based DNA detection.

DNA immobilization and detection using DNA binding proteins.

The immobilization of sensing bioreceptors is a critical feature affecting the final performance of a biosensor. For DNA detection, the (strept)avidin-biotin affinity interaction is often used for the immobilization of biotin-labeled oligonucleotides or PCR amplicons. Herein, DNA binding proteins are proposed as alternative universal anchors for both DNA immobilization and detection, based on the strong and specific affinity interaction between certain DNA binding proteins and their respective dsDNA binding sites. These binding sites can be incorporated in the target DNA molecule during synthesis and by PCR, eliminating the need for post-synthesis chemical modification and resulting in lower costs. When scCro DNA binding protein was immobilized on microplates and nitrocellulose membrane, both ssDNA and dsDNA targets were successfully detected. The detection limits achieved were similar to those obtained with the streptavidin-biotin system. However, the scCro system resulted in higher signals while using less amount of protein. The adsorption properties of scCro were superior to streptavidin's, making scCro a viable alternative as an anchor biomolecule for the development of DNA assays and biosensors. Finally, a nucleic acid lateral flow assay based solely on two different DNA binding proteins, scCro and dHP, was developed for the detection of a PCR amplicon. Overall, the proposed system appears to be very promising and with potential use for multiplex detection using various DNA binding proteins with different sequence specificities. Further work is required to better understand the adsorption properties of these biomolecules on nitrocellulose, optimize the assays comprehensively, and achieve improved sensitivities.

Electrochemical detection of alkaline phosphatase activity through enzyme-catalyzed reaction using aminoferrocene as an electroactive probe.

As a nonspecific phosphomonoesterase, alkaline phosphatase (ALP) plays a pivotal role in tissue mineralization and osteogenesis which is an important biomarker for the clinical diagnosis of bone and hepatobiliary diseases. Herein, we described a novel electrochemical method that used aminoferrocene (AFC) as an electroactive probe for the ALP activity detection. In the condition with imidazole and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), the AFC probe could be directly labeled on single-stranded DNA (ssDNA) by one-step conjugation. Specifically, thiolated ssDNA at 3'-terminals was modified to the electrode surface through Au-S bond. In the condition without ALP, AFC could be labeled on ssDNA by conjugating with phosphate groups. In the presence of ALP, phosphate groups were catalyzed to be removed from the 5'-terminal of ssDNA. The AFC probe cannot be labeled on ssDNA. Thus, the electrochemical detection of ALP activity was achieved. Under optimal conditions, the strategy presented a good linear relationship between current intensity and ALP concentration in the range of 20 to 100 mU/mL with the limit of detection (LOD) of 1.48 mU/mL. More importantly, the approach rendered high selectivity and satisfactory applicability for ALP activity detection. In addition, this method has merits of ease of operation, low cost, and environmental friendliness. Thus, this strategy presents great potential for ALP activity detection in practical applications. An easy, sensitive and reliable strategy was developed for the detection of alkaline phosphatase activity via electrochemical "Signal off".

Polystyrene Microparticles with Convergently Grown Mesoporous Silica Shells as a Promising Tool for Multiplexed Bioanalytical Assays.

Functional core/shell particles are highly sought after in analytical chemistry, especially in methods suitable for single-particle analysis such as flow cytometry because they allow for facile multiplexed detection of several analytes in a single run. Aiming to develop a powerful bead platform of which the core particle can be doped in a straightforward manner while the shell offers the highest possible sensitivity when functionalized with (bio)chemical binders, polystyrene particles were coated with different kinds of mesoporous silica shells in a convergent growth approach. Mesoporous shells allow us to obtain distinctly higher surface areas in comparison with conventional nonporous shells. While assessing the potential of narrow- as well as wide-pore silicas such as Mobil composition of matter no. 41 (MCM-41) and Santa Barbara amorphous material no. 15 (SBA-15), especially the synthesis of the latter shells that are much more suitable for biomolecule anchoring was optimized by altering the pH and both, the amount and type of the mediator salt. Our studies showed that the best performing material resulted from a synthesis using neutral conditions and MgSO4 as an ionic mediator. The analytical potential of the particles was investigated in flow cytometric DNA assays after their respective functionalization for individual and multiplexed detection of short oligonucleotide strands. These experiments revealed that a two-step modification of the silica surface with amino silane and succinic anhydride prior to coupling of an amino-terminated capture DNA (c-DNA) strand is superior to coupling carboxylic acid-terminated c-DNA to aminated core/shell particles, yielding limits of detection (LOD) down to 5 pM for a hybridization assay, using labeled complementary single-stranded target DNA (t-DNA) 15mers. The potential of the use of the particles in multiplexed analysis was shown with the aid of dye-doped core particles carrying a respective SBA-15 shell. Characteristic genomic sequences of human papillomaviruses (HPV) were chosen as the t-DNA analytes here, since their high relevance as carcinogens and the high number of different pathogens is a relevant model case. The title particles showed a promising performance and allowed us to unequivocally detect the different high- and low-risk HPV types in a single experimental run.

Gold nanoparticle-assisted plasmonic enhancement for DNA detection on a graphene-based portable surface plasmon resonance sensor.

The impact of different gold nanoparticle (GNP) structures on plasmonic enhancement for DNA detection is investigated on a few-layer graphene (FLG) surface plasmon resonance (SPR) sensor. Two distinct structures of gold nano-urchins (GNu) and gold nanorods (GNr) were used to bind the uniquely designed single-stranded probe DNA (ssDNA) of Mycobacterium tuberculosis complex DNA. The two types of GNP-ssDNA mixture were adsorbed onto the FLG-coated SPR sensor through the π-π stacking force between the ssDNA and the graphene layer. In the presence of complementary single-stranded DNA, the hybridization process took place and gradually removed the probes from the graphene surface. From SPR sensor preparation, the annealing process of the Au layer of the SPR sensor effectively enhanced the FLG coverage leading to a higher load of the probe DNA onto the sensing interface. The FLG was shown to be effective in providing a larger surface area for biomolecular capture due to its roughness. Carried out in the DNA hybridization study with the SPR sensor, GNu, with its rough and spiky structures, significantly reinforced the overall DNA hybridization signal compared with GNr with smooth superficies, especially in capturing the probe DNA. The DNA hybridization detection assisted by GNu reached the femtomolar range limit of detection. An optical simulation validated the extreme plasmonic field enhancement at the tip of the GNu spicules. The overall integrated approach of the graphene-based SPR sensor and GNu-assisted DNA detection provided the proof-of-concept for the possibility of tuberculosis disease screening using a low-cost and portable system to be potentially applied in remote or third-world countries.

Development of a Novel ssDNA Sequence for a Glycated Human Serum Albumin and Construction of a Simple Aptasensor System Based on Reduced Graphene Oxide (rGO).

Diabetes is one of the top 10 global causes of death. About one in 11 global adults have diabetes. As the disease progresses, the mortality rate increases, and complications can develop. Thus, early detection and effective management of diabetes are especially important. Herein, we present a novel glycated human serum albumin (GHSA) aptamer, i.e., GABAS-01, which has high affinity and specificity. The aptamer was selected by reduced graphene oxide-based systematic evolution of ligands by exponential enrichement (rGO-based SELEX) against GHSA. After five rounds of selection through gradually harsher conditions, GABAS-01 with high affinity and specificity for the target was obtained. GABAS-01 was labeled by FAM at the 5'-end and characterized by measuring the recovery of a fluorescence signal that is the result of fluorescence quenching effect of rGO. As a result, GABAS-01 had low-nanomolar Kd values of 1.748 ± 0.227 nM and showed a low limit of detection of 16.40 µg/mL against GHSA. This result shows the potential application of GABAS-01 as an effective on-site detection probe of GHSA. In addition, these properties of GABAS-01 are expected to contribute to detection of GHSA in diagnostic fields.

Spectrochemical Probing of MicroRNA Duplex Using Spontaneous Raman Spectroscopy for Biosensing Applications.

MicroRNAs are emerging as both diagnostic and therapeutic targets in different human pathologies. An accurate understanding of the structural dependency of microRNAs for their biological functions is essential for designing synthetic oligos with various base and linkage modifications that can transform into highly sensitive diagnostic devices and therapeutic molecules. In this proof-of-principle study, we have utilized label-free spontaneous Raman spectroscopy to understand the structural differences in sense and antisense microRNA-21 by hybridizing them with complementary RNA and DNA oligos. Overall, the results suggest that the changes in the Raman band at 785 cm-1 originating from the phosphodiester bond of the nucleic acid backbone, linking 5' phosphate of the nucleic acid with 3' OH of the other nucleotide, can serve as a marker to identify these structural variations. Our results support the application of Raman spectroscopy in discerning intramolecular (ssRNA and ssDNA) and intermolecular (RNA-RNA, RNA-DNA, and DNA-DNA hybrids) interactions of nucleic acids. This is potentially useful for developing biosensors to quantify microRNAs in clinical samples and to design therapeutic microRNAs with robust functionality.

Sperm chromatin condensation and single- and double-stranded DNA damage as important parameters to define male factor related recurrent miscarriage.

The aim of the present work is to characterize the relationship between sperm protamine deficiency and single- and double-stranded DNA damage and to assess the diagnostic potential of chromomycin A3 (CMA3). For that purpose, semen samples from 90 human males with different clinical features were included (fertile donors, patients with recurrent pregnancy loss [RPL], and infertile patients). DNA condensation was analyzed by CMA3 and different types of DNA fragmentation were analyzed through the comet assay. A positive correlation between DNA condensation and single-stranded DNA fragmentation was found (Rs = .456; p = .05). CMA3 presented differences between fertile donors and all other groups (p < .001). Interestingly, patients with RPL, who were able to achieve a pregnancy, and infertile patients showed similar values of CMA3 (p > .05). Receiver operating characteristic curves and the profiles obtained by the combination of Comet assays and CMA3 indicate that the CMA3 test may be an interesting approach to distinguish those subjects with higher pregnancy loss risk from fertile donors (CMA3 area under the curve 0.928, with a confidence interval of 0.849-1.000). The present work shows that DNA condensation is related to oxidative damage, which affects mainly protamine-rich regions. The profiles observed in different clinical groups showed that CMA3 might be useful for the diagnosis of RPL risk when combined with Comet assays.

Calcium-cation-doped polydopamine-modified 2D black phosphorus nanosheets as a robust platform for sensitive and specific biomolecule sensing.

Many polymer decorated/modified 2D nanomaterials have been developed as enhanced drug delivery systems and photothermal theranostic nanoagents. However, few reports describe the use of these novel nanomaterials as nanoplatforms for biomolecule sensing. Herein, we used calcium-cation-doped polydopamine-modified (PDA-modified) 2D black phosphorus (BP) nanosheets (BP@PDA) as a sensing nanoplatform for the detection of nucleic acids and proteins in complex biological samples. Fluorescent-dye-labeled single-strand DNA aptamer/probes are adsorbed by the Ca2+-doped BP@PDA mediated by calcium-cation coordination. The PDA coating enhances the stability of the inner BP, provides binding sites to DNA nucleobases, and quenches fluorescence. Without any chemical conjugation, this sensing nanoplatform selectively and specifically detects protein (human thrombin, linear range: 10-25 nM, detection limit: 0.02 nM), single-strand DNA (linear range: 1-10 nM, detection limit: 0.52 nM) in 1% serum diluted samples, and senses intracellular mRNAs (C-myc, and actin) in living cells. The nanoplatform exhibits the advantages of both the 2D nanomaterial (BP) and the coating polymer (PDA), naturally enters living cells unaided by transfection agents, resists enzymatic lysis and shows high biocompatibility. This nanoplatform design contributes towards future biomolecule analytical method development based on polymer decorated/modified 2D nanomaterials.

Evaluating digital PCR for the quantification of human nuclear DNA: determining target strandedness.

Digital polymerase chain reaction (dPCR) methodology has been asserted to be a "potentially primary" analytical approach for assigning DNA concentration. The essence of dPCR measurements is the independent dispersal of fragments into multiple reaction partitions, amplifying fragments containing a target nucleotide sequence until the signal from all partitions containing at least one such fragment rises above threshold, and then determining the proportion of partitions with an above-threshold signal. Should originally double-stranded DNA (dsDNA) fragments be converted into two single strands (ssDNA) prior to dispersal, the dPCR measurements could be biased high by as much as a factor of two. Realizing dPCR's metrological potential therefore requires analytical methods for determining the proportion of ssDNA in nominally dsDNA samples. To meet this need, we have investigated several approaches to this determination: A260 ratio, dPCR ratio, cdPCR staircase, and ddPCR enzyme. In our hands, only the endonuclease-based approach provides adequately accurate estimates for relatively small ssDNA proportions. We present four (enzyme, assay) pairs that provide self-consistent results for human nuclear DNA extracts. However, the proportion of ssDNA differs by as much as 50% between assays, apparently related to the guanine-cytosine (GC) content of the fragment near the assay's target sequence. While materials extracted by us have no more than 6% ssDNA content even after long storage, a commercially obtained PCR assay calibrant contains ≈18% ssDNA. Graphical abstract.