Protein-DNA Conjugates with a Discrete Number of Oligonucleotide Strands for Highly Reproducible Protein Quantification by the DNA Proximity Assay.

Protein-oligonucleotide conjugates are increasingly used as detection probes in biological applications such as proximity sensing and spatial biology. The preparation of high-quality conjugate probes as starting reagents is critical for achieving good and consistent performance, which we demonstrate via the DNA proximity assay (DPA) for the one-pot quantification of protein targets. We first established a complete conjugation and anion-exchange chromatography purification workflow to reproducibly obtain pure subpopulations of protein probes carrying a discrete number of oligonucleotide strands. A systematic study using the purified conjugate sub-populations confirmed that the order of conjugate (number of oligonucleotides per protein) and its purity (the absence of the unconjugated antibody) were important for ensuring optimal and reproducible assay performance. The streamlined workflow was then successfully used to conjugate a pair of universal DPA initiator oligonucleotides onto a wide range of binders including antibodies, nanobodies, and antigens which enabled the versatile detection of different types of proteins such as cytokines, total antibodies, and specific antibody isotypes. The good assay robustness (the inter-assay coefficient of variation lower than 5%) and linear calibration curve was achieved across all targets with just a single mix-and-incubate reaction step and a short reaction time of 30 min. We anticipate the streamlined protein-oligonucleotide probe preparation workflow developed in this work to have broad utility across applications leveraging the specificity of protein bio-recognition with the programmability of DNA hybridization.

Dynamically elongated associative toehold for tuning DNA circuit kinetics and thermodynamics.

Associative toehold is a powerful concept enabling efficient combinatorial computation in DNA circuit. A longer association length boosts circuit kinetics and equilibrium signal but results in higher leak rate. We reconcile this trade-off by using a hairpin lock design to dynamically elongate the effective associative toehold length in response to the input target. Design guidelines were established to achieve robust elongation without incurring additional leakages. Three hairpin initiators with different combinations of elongated associative toehold (4 → 6 nt, 5 → 8 nt and 6 → 9 nt) were shortlisted from the design framework for further discussion. The circuit performance improved in terms of reaction kinetics, equilibrium signal generated and limit of detection. Overall, the elongated associative toehold served as a built-in function to stabilize and favour the forward, desired reaction when triggered.

Dean migration of unfocused micron sized particles in low aspect ratio spiral microchannels.

We present an analysis of the microfluidic Dean migration of 2.5 µm particles, which do not meet focus criterion, in tall and low aspect ratio microchannels. We demonstrate the use of such low aspect ratio and tall spirals (h > 50 µm) for isolating high concentration (> 106 particles or cells/mL) micron sized particles without an initial off-chip dilution step. We specifically show the need for a sheath fluid for isolation and systematically analyze the particle stream profile (i.e. thickness and distance from the channel wall) as a function of downstream channel length and curvature ratio, with changes in the fluid velocity and the flow rate ratio of particles to sheath fluid (FRR). We also show that the width of the particle stream can control the particle migration and that a threshold stream width and Dean drag is necessary to initiate the particle stream migration from the channel wall. We then propose a design guide based on the selection of optimum curvatures, flow velocities and the FRRs required for achieving a narrow particle stream through a particular outlet. Finally, we use the design guide to demonstrate the isolation of bacteria from bladder epithelial cells.

Design of Split Proximity Circuit as a Plug-and-Play Translator for Point Mutation Discrimination.

Point mutations are a common form of genetic variation and have been identified as important disease biomarkers. Conventional methods for analyzing point mutations, e.g., polymerase chain reaction (PCR), are based on differences in thermal stability of the DNA duplex, which require extensive optimization of the reaction condition and nontrivial design of sequence-selective primers. This motivated the design of molecular translators to convert molecular inputs into generic output sequences, which allows for the target recognition and signal generation regions to be designed independently. In this work, we propose a translator design based on the concept of split proximity circuit (SPC) to achieve both high sequence selectivity and assay robustness using a universal reaction condition, i.e., room temperature and constant ionic concentration. We discussed the design aspects of the SPC recognition regions and demonstrated its plug-and-play capability to discriminate different point mutations for both DNA (seven G6PD mutations) and RNA (let-7 microRNA family members) targets while retaining the same signal generation region. Despite its simple design and nonstringent assay condition requirements, the SPC retained good analytical performance to detect subnanomolar target concentration within a reasonable time of an hour.

Dynamic Stabilization of DNA Assembly by Using Pyrrole-Imidazole Polyamide.

We used N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamide as an exogenous agent to modulate the formation of DNA assemblies at specific double-stranded sequences. The concept was demonstrated on the hybridization chain reaction that forms linear DNA. Through a series of melting curve analyses, we demonstrated that the binding of Py-Im polyamide positively influenced both the HCR initiation and elongation steps. In particular, Py-Im polyamide was found to drastically stabilize the DNA duplex such that its thermal stability approached that of an equivalent hairpin structure. Also, the polyamide served as an anchor between hairpin pairs in the HCR assembly, thus improving the originally weak interstrand stability. We hope that these proof-of-concept results can inspire future use of Py-Im polyamide as a molecular tool to modulate the formation of DNA assemblies.

The role of spacer sequence in modulating turn-on fluorescence of DNA-templated silver nanoclusters.

Guanine activation of fluorescence in DNA templated silver nanoclusters (AgNCs) is an interesting physical phenomenon which has yet to be fully understood to date. While the individual role of cytosine and guanine has been established, there is still a knowledge gap on how the AgNC-DNA system switches from dark to bright state. Here, we present evidence on the universal role of the DNA spacer sequence in physically separating two Ag+-binding cytosine sites to maintain the dark state while holding them together for structural re-organization by the guanine-rich strand to activate the bright state. The extent of turn-on signal could be modulated by adjusting the spacer length and composition. The ATATA spacer sequence was found to have negligible dark state fluorescence and a turn-on effect of 2440-fold, which was almost five times of the highest factor reported to date.

Localized Visualization and Autonomous Detection of Cell Surface Receptor Clusters Using DNA Proximity Circuit.

Cell surface receptors play an important role in mediating cell communication and are used as disease biomarkers and therapeutic targets. We present a one-pot molecular toolbox, which we term the split proximity circuit (SPC), for the autonomous detection and visualization of cell surface receptor clusters. Detection was powered by antibody recognition and a series of autonomous DNA hybridization to achieve localized, enzyme-free signal amplification. The system under study was the human epidermal growth factor receptor (HER) family, that is, HER2:HER2 homodimer and HER2:HER3 heterodimer, both in cell lysate and in situ on fixed whole cells. The detection and imaging of receptors were carried out using standard microplate scans and confocal microscopy, respectively. The circuit operated specifically with minimal leakages and successfully captured the receptor expression profiles on three cell types without any intermediate washing steps.

Engineering a robust DNA split proximity circuit with minimized circuit leakage.

DNA circuit is a versatile and highly-programmable toolbox which can potentially be used for the autonomous sensing of dynamic events, such as biomolecular interactions. However, the experimental implementation of in silico circuit designs has been hindered by the problem of circuit leakage. Here, we systematically analyzed the sources and characteristics of various types of leakage in a split proximity circuit which was engineered to spatially probe for target sites held within close proximity. Direct evidence that 3'-truncated oligonucleotides were the major impurity contributing to circuit leakage was presented. More importantly, a unique strategy of translocating a single nucleotide between domains, termed 'inter-domain bridging', was introduced to eliminate toehold-independent leakages while enhancing the strand displacement kinetics across a three-way junction. We also analyzed the dynamics of intermediate complexes involved in the circuit computation in order to define the working range of domain lengths for the reporter toehold and association region respectively. The final circuit design was successfully implemented on a model streptavidin-biotin system and demonstrated to be robust against both circuit leakage and biological interferences. We anticipate that this simple signal transduction strategy can be used to probe for diverse biomolecular interactions when used in conjunction with specific target recognition moieties.

Detection of G-Quadruplex Formation via Light Scattering of Defined Gold Nanoassemblies Modulated by Molecular Hairpins.

G-quadruplexes are of great scientific interest, as these unique DNA structures play key regulatory roles in cell replication, such as safeguarding against uncontrolled cellular divisions. The quadruplexes have also been applied for detecting DNA and protein biomarkers via methods like fluorescence resonance energy transfer (FRET) and gold nanoparticle (AuNP) aggregation. As an alternative and complementary platform to the established molecular techniques for the study of quadruplexes, we have developed a strategy coupling poly-G (PG)-mediated quadruplex formation with AuNP assembly detectable via dynamic light scattering (DLS). The presence of quadruplex-forming sequences also uniquely modifies the AuNP nanoassembly readout on DLS. In addition, molecular hairpins co-attached onto the AuNP together with PG successfully modulated the quadruplex-induced nanoassembly. Through molecular beacon-based fluorescence restoration and light scattering signal changes, the open/closed conformations of the hairpins are leveraged to tune the size of the quadruplex-mediated nanoassembly.

Gold Nanoplate-Based 3D Hierarchical Microparticles: A Single Particle with High Surface-Enhanced Raman Scattering Enhancement.

Formation of intended nano- and microstructures with regular building blocks has attracted much attention because of their potential applications in the fields of optics, electronics, and catalysis. Herein, we report a novel strategy to spontaneously grow three-dimensional (3D) hierarchical cabbagelike microparticles (CLMPs) constructed by individual Au nanoplates. By reducing gold precursor to gold atoms, N-(3-amidino)-aniline (NAAN) itself was oxidized to form poly(N-(3-amidino)-aniline) (PNAAN), which specifically binds on Au(111) facet as a capping agent and which leads to the formation of gold nanoplates. Because of the incomplete coverage of Au(111) facet, new gold nanoplate growth sites were spontaneously generated from the crystal plane of existing Au nanoplates for the growth of other nanoplates. This process continued until the nanoplate density reached its maximum range, eventually resulting in CLMPs with well-controlled structures. This opens a new avenue to utilize the imperfection during nanoparticle (NP) growth for the construction of microstructures. The individual CLMP shows excellent surface-enhanced Raman scattering (SERS) performance with high enhancement factor (EF) and good reproducibility as it integrates the SERS enhancement effects of individual Au nanoplate and the nanogaps formed by the uniform and hierarchical structures.

Engineering self-contained DNA circuit for proximity recognition and localized signal amplification of target biomolecules.

Biomolecular interactions have important cellular implications, however, a simple method for the sensing of such proximal events is lacking in the current molecular toolbox. We designed a dynamic DNA circuit capable of recognizing targets in close proximity to initiate a pre-programmed signal transduction process resulting in localized signal amplification. The entire circuit was engineered to be self-contained, i.e. it can self-assemble onto individual target molecules autonomously and form localized signal with minimal cross-talk. α-thrombin was used as a model protein to evaluate the performance of the individual modules and the overall circuit for proximity interaction under physiologically relevant buffer condition. The circuit achieved good selectivity in presence of non-specific protein and interfering serum matrix and successfully detected for physiologically relevant α-thrombin concentration (50 nM-5 µM) in a single mixing step without any further washing. The formation of localized signal at the interaction site can be enhanced kinetically through the control of temperature and probe concentration. This work provides a basic general framework from which other circuit modules can be adapted for the sensing of other biomolecular or cellular interaction of interest.

Detection of dissolved CO(2) based on the aggregation of gold nanoparticles.

A sensitive colorimetric assay of dissolved CO2 (dCO2) was developed based on the plasmon shift of gold nanoparticles (AuNPs). A water-soluble random copolymer poly(dimethyl acrylamide-co-(N-amidino)ethyl acrylamide), or P(DMA-co-NAEAA), containing amidine groups was synthesized. In the presence of dCO2, the amidine groups in the NAEAA block protonate and convert the polymer from a neutral to a positive-charged state, hence triggering the negative-charged AuNPs to aggregate by the electrostatic interaction. The degree of AuNP aggregation is dependent on the charge density of polymer, which is related to dCO2 concentration. The aggregation of AuNPs results in a red shift of the AuNP plasmonic spectrum, or a color change from red to blue. In addition, dCO2 concentration can be quantitatively measured by the UV absorbance change of the AuNP solution. A linear relationship between 0.264 and 6.336 hPa of dCO2 with a limit of detection (LOD) of 0.04 hPa can be acquired. This is the first report to detect dCO2 using the optical properties of nanoparticles.

Combination of in situ preconcentration and on-site analysis for phosphate monitoring in fresh waters.

Excess nutrients of phosphorus and nitrogen would lead to adverse impacts on a water body. It is important that their concentrations in a dynamic water ecosystem are measured accurately and constantly for an early warning before occurrences of algal blooms and for environmental management. Nevertheless, on-site measurements by existing technologies are often limited by the inherent sensitivities. In this study, a portable system for dissolved phosphate monitoring in freshwater based on the diffusive gradients in thin films (DGT) technique was developed. A polydiallydimethylammonium chloride (PDA) aqueous solution and a dialysis membrane were used as a binding phase and a diffusive layer in this preconcentration device, respectively. The binding properties of the PDA solution were evaluated in solutions of different pH (3 to 9) and varying concentrations of anions (2.0-20 mM). The amount of phosphates preconcentrated in the devices was measured by ultraviolet-visible (UV) spectroscopy to obtain the concentrations in waters without elution steps. The devices were validated in synthetic river water with good agreement with the theoretical prediction and in natural river water. A system combining this preconcentration device and a compact detection chamber equipped with a pair of light emitting diodes (LED) was studied in lab synthetic solutions for on-site monitoring of phosphate concentrations and their fluctuations.

Gold nanostructures for the multiplex detection of glucose-6-phosphate dehydrogenase gene mutations.

We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.

One-Pot Detection of Proteins Using a Two-Way Extension-Based Assay with Cas12a.

Protein biomarkers are an important class of biomarkers in disease diagnosis and are traditionally detected by enzyme-linked immunosorbent assay and mass spectrometry, which involve multiple steps and a complex workflow. In recent years, many CRISPR-Cas12a-based methods for protein detection have been developed; however, most of them have not overcome the workflow complications observed in traditional assays, limiting their applicability in point-of-care testing. In this work, we designed a single-step, one-pot, and proximity-based isothermal immunoassay integrating CRISPR Cas12a for homogeneous protein target detection with a simplified workflow and high sensitivity. Probes consisting of different binders (small molecule, aptamer, and antibody) conjugated with oligonucleotides undergo two-way extension upon binding to the protein targets, leading to downstream DNA amplification by a pair of nicking enzymes and polymerases to generate target sequences for Cas12a signal generation. We used the streptavidin-biotin model to demonstrate the design of our assay and proved that all three elements of protein detection (target protein binding, DNA amplification, and Cas12a signal generation) could coexist in one pot and proceed isothermally in a single buffer system at a low reaction volume of 10 µL. The plug-and-play applicability of our assay has been successfully demonstrated using four different protein targets, streptavidin, PDGF-BB, antidigoxigenin antibody, and IFNγ, with the limit of detection ranging from fM to pM.

Protein-to-DNA Converter with High Signal Gain.

DNA isothermal amplification techniques have been applied extensively for evaluating nucleic acid inputs but cannot be implemented directly on other types of biomolecules. In this work, we designed a proximity activation mechanism that converts protein input into DNA barcodes for the DNA exponential amplification reaction, which we termed PEAR. Several design parameters were identified and experimentally verified, which included the choice of enzymes, sequences of proximity probes and template strand via the NUPACK design tool, and the implementation of a hairpin lock on the proximity probe structure. Our PEAR system was surprisingly more robust against nonspecific DNA amplification, which is a major challenge faced in existing formats of the DNA-based exponential amplification reaction. The as-designed PEAR exhibited good target responsiveness for three protein models with a dynamic range of 4-5 orders of magnitude down to femtomolar input concentration. Overall, our proposed protein-to-DNA converter module led to the development of a stable and robust configuration of the DNA exponential amplification reaction to achieve high signal gain. We foresee this enabling the use of protein inputs for more complex molecular evaluation as well as ultrasensitive protein detection.

High-resolution 3D imaging and quantification of gold nanoparticles in a whole cell using scanning transmission ion microscopy.

Increasing interest in the use of nanoparticles (NPs) to elucidate the function of nanometer-sized assemblies of macromolecules and organelles within cells, and to develop biomedical applications such as drug delivery, labeling, diagnostic sensing, and heat treatment of cancer cells has prompted investigations into novel techniques that can image NPs within whole cells and tissue at high resolution. Using fast ions focused to nanodimensions, we show that gold NPs (AuNPs) inside whole cells can be imaged at high resolution, and the precise location of the particles and the number of particles can be quantified. High-resolution density information of the cell can be generated using scanning transmission ion microscopy, enhanced contrast for AuNPs can be achieved using forward scattering transmission ion microscopy, and depth information can be generated from elastically backscattered ions (Rutherford backscattering spectrometry). These techniques and associated instrumentation are at an early stage of technical development, but we believe there are no physical constraints that will prevent whole-cell three-dimensional imaging at <10 nm resolution.

Conformal growth of nanocrystalline CdX (X = S, Se) on mesoscopic NiO and their photoelectrochemical properties.

Semiconductor-sensitized NiO photocathodes have been fabricated by successive ionic-layer adsorption and reaction (SILAR) deposition of CdS, CdSe and cascaded CdS/CdSe onto mesoscopic NiO films. Detailed morphological and structural characterization reveals that the growth of CdS and CdSe on mesoscopic NiO electrodes results in the formation of crystalline and conformal layers under ambient conditions. With a polysulfide redox electrolyte and a Pt counter electrode, CdX (X = S and Se)-sensitized p-NiO solar cells operating in a photocathodic mode are unambiguously demonstrated when NiO blocking layers are used, which are critical to prevent anodic photocurrent due to electron injection from CdX into the SnO2:F substrate. To decrease the recombination rate, a CdS barrier layer was deposited between NiO and a CdSe sensitizer which results in much enhanced cell performance. Front and rear spectral incident photon-to-current efficiency (IPCE) measurements were used to investigate charge collection and separation in the cells. The measurements indicate that charge collection in this system is limited by a short hole diffusion length.

Design strategies for countering the effect of fluorophore-quencher labelling on DNA hairpin thermodynamics.

We report the impact of fluorophore-quencher labelling on the thermodynamics of hairpin opening by testing five fluorophores and two quenchers labelled at the end and/or internal positions. Two counter strategies were introduced, i.e. label the hairpin probe at an internal position or append an external hairpin stem on the trigger strand to promote coaxial stacking hybridization. The observations remained valid for complex hairpin opening operations such as hybridization chain reaction.

Harnessing the Immunogenic Potential of Gold Nanoparticle-Based Platforms as a Therapeutic Strategy in Breast Cancer Immunotherapy: A Mini Review.

Breast cancer remains the most common malignancy among women worldwide. Although the implementation of mammography has dramatically increased the early detection rate, conventional treatments like chemotherapy, radiation therapy, and surgery, have significantly improved the prognosis for breast cancer patients. However, about a third of treated breast cancer patients are known to suffer from disease recurrences and progression to metastasis. Immunotherapy has recently gained traction due to its ability to establish long-term immune surveillance, and response for the prevention of disease recurrence and extension of patient survival. Current research findings have revealed that gold nanoparticles can enhance the safety and efficacy of cancer immunotherapy, through their unique intrinsic properties of good biocompatibility, durability, convenient surface modification, as well as enhanced permeability and retention effect. Gold nanoparticles are also able to induce innate immune responses through the process of immunogenic cell death, which can lead to the establishment of lasting adaptive immunity. As such gold nanoparticles are considered as good candidates for next generation immunotherapeutic strategies. This mini review gives an overview of gold nanoparticles and their potential applications in breast cancer immunotherapeutic strategies.