Ratiometric fluorescence sensing NADH using AIE-dots transducers at the point of care.

Reduced nicotinamide adenine dinucleotide (NADH) has a strong impact on physiological metabolism, and its concentration is related to metabolic and neurodegenerative diseases. A more reliable and accurate detection method for NADH quantitation is needed for early disease diagnosis and point-of-care testing. Aggregation-induced emission (AIE) materials are widely used to improve the sensitivity in analytes assays due to their anti-aggregation-caused quenching property. Here we developed TPA-BQD-Py AIE-dots transducers and evaluated its performance in NADH detection. The NADH concentration-dependent ratiometric sensing was based on electron transfer from TPA-BQD-Py AIE-dots to NADH with variable fluorescence intensity at 584 nm and 470 nm, resulting in high sensitivity (limit of detection at 110 nM), photostability, selectivity, and a rapid and reversible response. We further developed the application of TPA-BQD-Py AIE-dots transducers in in vivo NADH imaging using a smartphone and digital camera, respectively, demonstrating the potential for NADH point-of-care testing.

Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection.

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

AND-Logic Cascade Rolling Circle Amplification for Optomagnetic Detection of Dual Target SARS-CoV-2 Sequences.

DNA logic operations are accurate and specific molecular strategies that are appreciated in target multiplexing and intelligent diagnostics. However, most of the reported DNA logic operation-based assays lack amplifiers prior to logic operation, resulting in detection limits at the subpicomolar to nanomolar level. Herein, a homogeneous and isothermal AND-logic cascade amplification strategy is demonstrated for optomagnetic biosensing of two different DNA inputs corresponding to a variant of concern sequence (containing spike L452R) and a highly conserved sequence from SARS-CoV-2. With an "amplifiers-before-operator" configuration, two input sequences are recognized by different padlock probes for amplification reactions, which generate amplicons used, respectively, as primers and templates for secondary amplification, achieving the AND-logic operation. Cascade amplification products can hybridize with detection probes grafted onto magnetic nanoparticles (MNPs), leading to hydrodynamic size increases and/or aggregation of MNPs. Real-time optomagnetic MNP analysis offers a detection limit of 8.6 fM with a dynamic detection range spanning more than 3 orders of magnitude. The accuracy, stability, and specificity of the system are validated by testing samples containing serum, salmon sperm, a single-nucleotide variant, and biases of the inputs. Clinical samples are tested with both quantitative reverse transcription-PCR and our approach, showing highly consistent measurement results.

On-Particle Hyperbranched Rolling Circle Amplification-Scaffolded Magnetic Nanoactuator Assembly for Ferromagnetic Resonance Detection of MicroRNA.

Inspired by natural molecular machines, scientists are devoted to designing nanomachines that can navigate in aqueous solutions, sense their microenvironment, actuate, and respond. Among different strategies, magnetically driven nanoactuators can easily be operated remotely in liquids and thus are valuable in biosensing. Here we report a magnetic nanoactuator swarm with rotating-magnetic-field-controlled conformational changes for reaction acceleration and target quantification. By grafting nucleic acid amplification primers, magnetic nanoparticle (MNP) actuators can assemble and be fixed with a flexible DNA scaffold generated by surface-localized hyperbranched rolling circle amplification in response to the presence of a target microRNA, osa-miR156. Net magnetic anisotropy changes of the system induced by the MNP assembly can be measured by ferromagnetic resonance spectroscopy as shifts in the resonance field. With a total assay time of ca. 120 min, the proposed biosensor offers a limit of detection of 6 fM with a dynamic detection range spanning 5 orders of magnitude. The specificity of the system is validated by testing different microRNAs and salmon sperm DNA. Endogenous microRNAs extracted from Oryza sativa leaves are tested with both quantitative reverse transcription-PCR and our approach, showing comparable performances with a Pearson correlation coefficient >0.9 (n = 20).

Apex-Confined Plasmonic Tip for High Resolution Tip-Enhanced Raman Spectroscopic Imaging of Carbon Nanotubes.

This paper reports a handy technical scheme to decorate atomic force microscopy (AFM) tips toward tip-enhanced Raman spectroscopy (TERS) applications. The major attraction of these homemade tips lies in that silver decoration can be confined at the apex of commercial tips by the means of an AFM-controlled electrochemical reaction. The reduction of Ag+ occurs in a highly sealed environment to secure the metal coating efficiency. Key factors include silver nitrate solution to provide Ag+, ambient relative humidity and temperature in a humidity cell, electric potential bias, and tip-surface distance. Subsequently, these silver-coated tips are evaluated for TERS measurement of carbon nanotubes (CNTs) so that both morphological and chemical characteristics of CNTs are concurrently obtained. The Raman spectra reveal that our plasmonic tip competently possesses an ∼30-fold local field signal increase and the corresponding TERS image laterally resolves at the single-pixel level.

Calling the amino acid sequence of a protein/peptide from the nanospectrum produced by a sub-nanometer diameter pore.

The blockade current that develops when a protein translocates across a thin membrane through a sub-nanometer diameter pore informs with extreme sensitivity on the sequence of amino acids that constitute the protein. The current blockade signals measured during the translocation are called a nanospectrum of the protein. Whereas mass spectrometry (MS) is still the dominant technology for protein identification, it suffers limitations. In proteome-wide studies, MS identifies proteins by database search but often fails to provide high protein sequence coverage. It is also not very sensitive requiring about a femtomole for protein identification. Compared with MS, a sub-nanometer diameter pore (i.e. a sub-nanopore) directly reads the amino acids constituting a single protein molecule, but efficient computational tools are still required for processing and interpreting nanospectra. Here, we delineate computational methods for processing sub-nanopore nanospectra and predicting theoretical nanospectra from protein sequences, which are essential for protein identification.

Measurements of the size and correlations between ions using an electrolytic point contact.

The size of an ion affects everything from the structure of water to life itself. In this report, to gauge their size, ions dissolved in water are forced electrically through a sub-nanometer-diameter pore spanning a thin membrane and the current is measured. The measurements reveal an ion-selective conductance that vanishes in pores <0.24 nm in diameter-the size of a water molecule-indicating that permeating ions have a grossly distorted hydration shell. Analysis of the current noise power spectral density exposes a threshold, below which the noise is independent of current, and beyond which it increases quadratically. This dependence proves that the spectral density, which is uncorrelated below threshold, becomes correlated above it. The onset of correlations for Li+, Mg2+, Na+ and K+-ions extrapolates to pore diameters of 0.13 ± 0.11 nm, 0.16 ± 0.11 nm, 0.22 ± 0.11 nm and 0.25 ± 0.11 nm, respectively-consonant with diameters at which the conductance vanishes and consistent with ions moving through the sub-nanopore with distorted hydration shells in a correlated way.

Method for Dynamically Detecting Secretions from Single Cells Using a Nanopore.

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.

Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore.

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (i.e., a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of a single protein molecule by measuring volumes of 0.1 nm3 per read.

Single-molecule protein identification by sub-nanopore sensors.

Recent advances in top-down mass spectrometry enabled identification of intact proteins, but this technology still faces challenges. For example, top-down mass spectrometry suffers from a lack of sensitivity since the ion counts for a single fragmentation event are often low. In contrast, nanopore technology is exquisitely sensitive to single intact molecules, but it has only been successfully applied to DNA sequencing, so far. Here, we explore the potential of sub-nanopores for single-molecule protein identification (SMPI) and describe an algorithm for identification of the electrical current blockade signal (nanospectrum) resulting from the translocation of a denaturated, linearly charged protein through a sub-nanopore. The analysis of identification p-values suggests that the current technology is already sufficient for matching nanospectra against small protein databases, e.g., protein identification in bacterial proteomes.

Reading the primary structure of a protein with 0.07 nm3 resolution using a subnanometre-diameter pore.

The primary structure of a protein consists of a sequence of amino acids and is a key factor in determining how a protein folds and functions. However, conventional methods for sequencing proteins, such as mass spectrometry and Edman degradation, suffer from short reads and lack sensitivity, so alternative approaches are sought. Here, we show that a subnanometre-diameter pore, sputtered through a thin silicon nitride membrane, can be used to detect the primary structure of a denatured protein molecule. When a denatured protein immersed in electrolyte is driven through the pore by an electric field, measurements of a blockade in the current reveal nearly regular fluctuations, the number of which coincides with the number of residues in the protein. Furthermore, the amplitudes of the fluctuations are highly correlated with the volumes that are occluded by quadromers (four residues) in the primary structure. Each fluctuation, therefore, represents a read of a quadromer. Scrutiny of the fluctuations reveals that the subnanometre pore is sensitive enough to read the occluded volume that is related to post-translational modifications of a single residue, measuring volume differences of ∼0.07 nm3, but it is not sensitive enough to discriminate between the volumes of all twenty amino acids.

Determination of mechanical properties of soft tissue scaffolds by atomic force microscopy nanoindentation.

While the determination of mechanical properties of a hard scaffold is relatively straightforward, the mechanical testing of a soft tissue scaffold poses significant challenges due in part to its fragility. Here, we report a new approach for characterizing the stiffness and elastic modulus of a soft scaffold through atomic force microscopy (AFM) nanoindentation. Using collagen-chitosan hydrogel scaffolds as model soft tissue scaffolds, we demonstrated the feasibility of using AFM nanoindentation to determine a force curve of a soft tissue scaffold. A mathematical model was developed to ascertain the stiffness and elastic modulus of a scaffold from its force curve obtained under different conditions. The elastic modulus of a collagen-chitosan (80%/20%, v/v) scaffold is found to be 3.69 kPa. The scaffold becomes stiffer if it contains more chitosan. The elastic modulus of a scaffold composed of 70% collagen and 30% chitosan is about 11.6 kPa. Furthermore, the stiffness of the scaffold is found to be altered significantly by extracellular matrix deposited from cells that are grown inside the scaffold. The elastic modulus of collagen-chitosan scaffolds increased from 10.5 kPa on day 3 to 63.4 kPa on day 10 when human foreskin fibroblast cells grew inside the scaffolds. Data acquired from these measurements will offer new insights into understanding cell fate regulation induced by physiochemical cues of tissue scaffolds.