Abstract
Point-of-Care Testing
Conformation-Sensitive Antibody-Based Point-of-Care Immunosensor for Serum Ca2+ Using Two-Dimensional Sequential Binding Reactions
Calcium (II) ions play a critical role in blood coagulation and act as a cofactor for a broad range of enzymes. Current methods that assess blood calcium include atomic absorption and electrochemical detection that require expensive instruments and careful maintenance. Spectrophotometric detection is another widely used method due to its simplicity. However, it measures total blood calcium; thus, it is limited by the need to estimate albumin titer in serum, as well as the influence of unknown factors on ionic calcium levels.
To address such needs, an immunoanalytical system to quantify free ionic calcium has been developed for point-of-care applications. This system utilizes a sequential binding reaction system involving Ca2+ and calcium binding protein (CBP) before inducing antigen–antibody binding. CBP is labeled with colloidal gold using a biotin–streptavidin linkage, and the antibody is immobilized on the membrane surface. In the absence of Ca2+, this antibody binding does not occur.
To rapidly and accurately analyze the quantity of binding, a two-dimensional (2D) chromatography cross-flow system (horizontal and vertical) is employed to generate a signal proportional to the analyte concentration. This format minimizes retarded flow of the gold tracer in high-calcium titer samples. This signal is then captured on a smartphone before integrating the area under the analyte line curve to obtain a signal value for each sample. Further work will involve the development of mobile applications for signal quantification, as well as incorporating the detection of other analytes (e.g., parathyroid hormone) (Park, J.; et al. Biosens. Bioelectron.
Sustained-Release Synthetic Biomarkers for Monitoring Thrombosis and Inflammation Using Point-of-Care Compatible Readouts
Within hospital settings, postoperative patients are monitored for prothrombin fragment 1.2 and D-dimer, which serve as biomarkers for thrombosis or evidence of pathogenic infection. In contrast, currently discharged patients are merely educated regarding symptoms of potential complications. Monitoring such at-risk patients following discharge significantly decreases morbidity, mortality, and the ensuing costs of such complications. Previously, Dudani and colleagues developed molecular tests for monitoring disease status by measuring synthetic biomarkers (e.g., protease activity from thrombin in blood clots).
To effectively monitor postoperative discharged patients, they adapted their previous design into a novel point-of-care diagnostic system that can be administrated subcutaneously upon hospital discharge and then enters the bloodstream and exhibits sustained-release kinetics (from hours to months).
Large (40 kDa) poly(ethylene glycol) (PEG) scaffolds are equipped with protease-sensitive reporters for thrombin (PEG-T1E/Q) and MMP9 (PEG-M1E/Q). Compared with 20 kDa PEG and iron oxide nanoparticles, the 40 kDa PEG exhibits improved blood plasma retention with a half-life exceeding 20 h. After establishing pharmacokinetics of this diagnostic system, the assay is utilized on a mouse model of thrombosis where blood clots are generated in the lung vasculature by intravenous (IV) injection of thromboplastin (TP). Although PEG-T1E simultaneously delivered intravenously with TP effectively generates a positive urinary signal, it is not representative of realistic clinical scenarios. Instead, offset diagnostic administration (2 h prior) demonstrates positive diagnostic reporting ability for subcutaneous (SC) but not IV administration. Using a model of mice infected with pulmonary delivered bacteria (Pseudomonas aeruginosa), PEG-M1E exhibits a significantly higher signal 24 h postinfection compared with early time periods (2 h).
Finally, the study demonstrates the development of a companion paper–based enzyme-linked immunosorbent assay (ELISA) to diagnose thrombosis (SC administration) from the urine of infected mice. The development of such diagnostics through SC implantation (facilitating sustained release) and facile urinary readouts enable long-term monitoring of postoperative patients (Dudani, J. S.; et al. Adv. Funct. Mater.
Instrument-Free Point-of-Care Molecular Detection of Zika Virus
Zika virus (ZIKV) infections have been highly prioritized by the World Health Organization (WHO) because they are linked to a number of severe neurological defects in newborns following transmission from infected mothers. ZIKV-infected individuals are difficult to identify, as infected individuals are often asymptomatic or present symptoms common to other less devastating diseases. Current detection assays for ZIKV include lateral-flow immune-chromatographic assays or reverse-transcription (RT)–PCR (gold standard). Both methods have limitations, including low sensitivity/specificity, and they require significant experimental resources (e.g., sample preparation, expensive equipment, and trained personnel). Such limitations are further exacerbated in resource-poor countries.
Under these constraints, the authors design a reverse-transcription loop-mediated amplification (RT-LAMP) diagnostic for ZIKV. A set of six primers are chosen on the basis of high homology among ZIKV isolates, as well as high divergence from other flaviviruses. To facilitate point-of-care (POC) diagnosis, a custom-made disposable microfluidic cassette combines the isolation of viral nucleic acid (from saliva samples) with its isothermal amplification and detection. The cassette is placed in an electricity-free cup that generates heat (from exothermic reactions). Detection of amplicon double-stranded DNA is achieved using colorless leucocrystal violet (LCV). A commercially available flameless ration heater (FRH) produces heat when the magnesium alloy interacts with water maintaining temperature at 63 °C, and each test is estimated to cost $2.
As a proof of concept, ZIKV is spiked in saliva containing various concentrations of virus (0, 5, 50, and 500 PFU/mL) at an ambient temperature of 18 °C. The microfluidic cassette is inserted into the heated cup and activated after water is added. After 40 min, the RT-LAMP reaction is complete and the saliva, spiked with ZKIV, displays a violet color. In contrast, the negative control remains colorless as there is an absence of amplified dsDNA products. Furthermore, the on-chip tests are validated against conventional RT-LAMP benchtop tests with similar results.
Future iterations of this POC platform will substitute the LCV dye with intercalating DNA dyes and interface with smartphones. Fluorescence emission can be recorded during the amplification process following flashlight excitation and potentially include geotagging features. Other advances may include custom-made applications to analyze data and estimate template concentration. This platform may further be multiplexed to detect other mosquito pathogens (e.g., dengue and chikungunya) and is highly viable for diagnosis in resource-poor developing nations (Song, J.; et al. Anal. Chem.
Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation
HIV drug-resistant (DR) strains arise due to antiretroviral drug levels falling below therapeutic levels. In the absence of DR screening, DR HIV spreading reduces the potency of first-line antiretroviral treatment. Sanger (consensus) sequencing is the standard method to screen for HIV-DR. However, detection is limited with smaller proportions of HIV-DR (<15%−30%). Although Sanger sequencing simultaneously assesses the genotype over the entire HIV pol region, DR screening can be achieved by assessing a smaller set of specific single-nucleotide mutations associated with antiretroviral drug resistance.
The oligonucleotide ligation assay (OLA) has been developed and validated to detect multiple HIV mutations associated with resistance. It takes advantage of probe hybridization and ligation specificity to identify specific base mutations. Following probe ligation, the probes are detected via surface capture, denaturation of oligonucleotide from target DNA, and enzyme-based detection (CDD). Using probes specific for wild-type and mutant genotypes, estimates can be made for the relative proportion of HIV-DR to a standard comparable to sequencing methods. While being comparatively cost-effective with a higher sensitivity, its duration and complexity hinders its transfer to resource-limited laboratories. This CDD procedure is adapted to a paper strip format to reduce the number of steps and assay time and allow visual detection of results.
Standard plate assays (plate) use streptavidin-coated well plates to capture ligation probes and denaturing buffers to remove nonligated probes from template DNA. On the other hand, paper OLA streptavidin (paper) is immobilized onto nitrocellulose strips via spotting and drying. The analytical ability of paper CDD is compared with the plate version with a plasmid mixture dilution series for the Y181C mutation. The paper assay successfully detects the different proportions of mutations even though there are differences in the extent of assay saturation point.
Clinical specimens are tested next. At low % MUT (mutant), the assay shows linear correlation between both formats, while signal saturation is observed for the paper assay at higher % MUT. Overall, good correlation is observed between both formats. The OLA allows identification of HIV drug resistance at a lower cost and higher sensitivity than Sanger sequencing, although the complexity of the protocol is a bottleneck in its implementation. The simplification of the detection protocol (time and complexity) presented here could allow easier transfer of the OLA to laboratories with limited resources (Panpradist, N.; et al. PLoS ONE
Clinical Automation
Automated Scoring of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus by Use of WASPLab Image Analysis Software
Automation benefits clinical microbiology by reducing turnaround time and labor costs and improving patient care. Features such as conveyor/track systems, programmable software, and digital cameras have been used for imaging plates with the goal to create fully automated systems that process specimens, incubate plates, image plates for interpretation, and pick colonies for further culture processing. Kiestra total laboratory automation and WASPLab are two such commercially available examples.
Chromogenic agars are culture substrates that utilize differences in pathogen metabolism to create enzymatic reactions specific for target organisms such as vancomycin-resistant enterococci (VRE), group B streptococcus (GBS), and methicillin-resistant Staphylococcus aureus (MRSA). In the presence of their target organisms, secreted factors interact with the chromogen to produce pigmentation (mauve, pink, or green). As such, digital imaging software can distinguish color differences and be used to set color thresholds for detecting target organisms.
WASPLab chromogenic detection module (CDM) is a software that analyzes digital images and converts red-green-blue (RGB) images into three-dimensional (3D) spaces composed of hue, saturation, and value (HSV) to identify nonnegative media plates. The software looks for the selected color pattern within the specified tolerance, with those within defined parameters marked as nonnegative, but negative if no pixel contains an HSV score outside these parameters. Thus, the authors hypothesize that WASPLab can accurately sort chromogenic MRSA plates into negative and nonnegative as a means of detecting organism presence.
A multisite evaluation to identify MRSA from swab cultures plated on various chromogenic agars is performed using WASPLab. These are compared directly to manual reading for positive MRSA detection. To demonstrate robustness and accuracy of CDM software, three chromogenic media are tested: MRSASelect, chromID MRSA, and BD CHROMagar MRSA. All three have different pigmentations, and hence specific imaging thresholds are set for each agar type. The prevalence of MRSA at the test sites is found to be 2.37%. An additional 5210, or 9%, plates are manually read as negative (by laboratory technicians) but reported as nonnegative by the CDM software. Crucially, the software does not read manual-positive plates as negative. The overall sensitivity of the detection method is 100%, with 90.7% specificity.
There was a further analysis of the manual-negative/automation-positive plates. Among these, the failure for detection could be categorized as the following: automation positive/second manual positive (indicating technician error), residual matrix, or borderline colors relating to the set threshold. Importantly, there were no manual-positive/automation-negative samples, meaning that automated detection could be utilized for a quick removal of negative plates. In turn, this would ease the burden of large-volume screens and improve microorganism detection efficiency. Thus, this is the first high-volume, multisite study demonstrating full lab automation to perform image analysis to identify MRSA. Promisingly, it was found to have greater detection sensitivity than manual observation techniques (Faron, M. L.; et al. J. Clin. Microbiol.
Toward Biomarker Development in Large Clinical Cohorts: An Integrated High-Throughput 96-Well Plate–Based Sample Preparation Workflow for Versatile Downstream Proteomic Analyses
Precision medicine has been a driver of novel clinical proteomic techniques to quantify proteins from large sample cohorts. Simple, reproducible, and cost-effective preparation routines are required to profile such a large number of samples. In addition, these need to accommodate 96-well plate formats for universal practice and be able to be modified readily for automation.
To date, the widely adopted filter-assisted sample processing (FASP) methods are carried out using 10 or 30 kDa spin filter units with the addition of multiple reagents and prolonged centrifugation, making it suitable to process only a small number of samples. Attempts to develop high-throughput preparation methods for 96 wells all require specially designed or modified apparatus. The bottleneck in sample preparation methods is high-speed centrifugation of the protein precipitates since 96-well plates can only withstand 2000g. Such high-speed centrifugation is challenging to incorporate into sample automation platforms.
In this report, the authors have developed a straightforward and robust protocol via protein precipitation with acetone and centrifugation at slow speed in regular 96-well plates (96PACS). Using complex protein mixtures, it is demonstrated that the 96PACS method provides higher proteome coverage and reproducibility than FASP. These are then tested using liquid chromatography–tandem mass spectrometry (LC-MSMS) experiments using data-independent acquisition (DIA) and parallel reaction monitoring (PRM) methods with minimal variation in the quality of analysis. Also, the 96PACS method does not lead to the depletion of proteins or preferential enrichment of other species.
Thereafter, this technique is modified for automation and used to identify serum markers for predicting liver fibrosis and necroinflammation at early stages in patients with chronic hepatitis B viral infections. The difficulty in proteomic identification of early-stage serum biomarkers is their low concentration levels. Serum protein signatures from 12 CHB patients are compared with those of 12 healthy controls using 96PACS and analyzed using LC-MSMS. Further unsupervised clustering statistical analysis showed a distinct signature between the two groups. Through the analysis, three biomarkers could be identified as early markers of liver necroinflammation in CHB patients: alpha-2-macroglobulin, apolipoprotein B, and complement component 4B.
There is particular interest in identifying biomarkers for early detection of diseases, especially in asymptomatic stages before disease progression. A major hurdle in biomarker identification is the low biomarker concentration compared with advanced stages of the disease. Because of its ease of use, robustness, and adaptability to existing sample processing workflows, the 96PACS technique can aid in the development of inexpensive, high-throughput, and flexible clinical applications for proteomics (Sun, Z.; et al. Anal. Chem.
Nanomedicine
Light-Responsive Biodegradable Nanomedicine Overcomes Multidrug Resistance via NO-Enhanced Chemosensitization
Multidrug resistance (MDR) is a challenging issue in cancer therapy because it can cause chemotherapy to fail or require increased effective drug dosage. MDR occurs because of the overexpression of transmembrane efflux pumps that eliminate exogenous substrates out of cells. One example is the ATP-binding cassette (ABC) transporter, which is overly expressed in the hypoxic and the highly oxidative tumor environment. Moreover, cancers with increased hypoxia and excessive reactive oxygen species (ROS) prognose increased mortality. Within this context, reducing agents counter the effects of MDR; nitric oxide (NO) unsurprisingly reverses MDR and restores chemotherapeutic efficacy. NO, a highly diffusible and short-lived radical, reacts with ROS in tumor tissue and deactivates transport proteins.
To develop a deliverable therapeutic, BNN6 (an NO donor sensitive to UV illumination) and doxorubicin (DOX) are loaded into mono-methoxy(polyethylene glycol)-poly(lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles. Under physiological conditions, the nanoparticles are stable, but they release NO and DOX after UV irradiation. Following the activation of nanoparticles with UV light for 2 min, they collapse and release NO and DOX.
In MDR cells (OVCAR-8/ADR), an intracellular NO probe (RBSP) successfully detected the release of NO to validate the particle’s light-responsive behavior. Following 48 h treatment with the light-responsive nanoparticles, fluorescent DOX accumulates in the nucleus and MDR cell growth is significantly inhibited. In comparison, the untriggered particles group accumulates most of its signal in the cytoplasm and the free DOX group only expresses minimal intracellular signal. Correspondingly, the UV-triggered particles generate the greatest amount of cytotoxicity with an IC50 value of ~20 µg/mL. This nanoparticle is expected to prolong blood circulation and trigger NO/DOX release as a novel anticancer therapy (Fan, J.; et al. ACS Appl. Mater. Interfaces
Self-Assembled RNA Triple-Helix Hydrogel Scaffold for microRNA Modulation in the Tumor Microenvironment
The full potential of using microRNA (miRNA) to suppress cancer is hampered by the lack of suitable delivery vehicles for efficient delivery. Issues such as poor miRNA in vivo stability, nonspecific biodistribution, and other less desirable side effects can be overcome by local and sustained delivery platforms. Current methods utilizing nanoparticles exhibit miRNA dissociation from the vehicle, poor stability, and suboptimal targeting, all of which contribute to poor efficacy.
To circumvent these issues, self-assembled three-dimensional (3D) oligonucleotide structures (RNA triple helix) are considered for miRNA therapeutic delivery in oncology. Their advantages include potentially higher stability and transfection cancer cell efficiency. Structurally, a triple helix is formed by adding a small synthetic ssRNA (antagomiR) to a miRNA duplex (miR-205), which is bound by Watson–Crick and Hoogsteen hydrogen bonds.
To gauge its efficacy, the triple helix is tested on an orthotopic triple-negative breast cancer (TNBC) mouse model lacking progesterone, estrogen, and HER2 receptors. TNBC is not responsive to hormone or HER2-targeted therapies—making it suitable for gene therapy approaches. By delivering antagomiRs that sterically hinder miRNAs that aid tumor progression and miR-205 mimics that promote tumor suppression, Conde et al. hypothesized that TNBC could be suppressed using a twin-pronged miRNA approach.
The RNA triple helices are combined with dendrimer nanoparticles that form a hydrogel upon mixing with dextran aldehyde. To report in vivo release following hybridization with complementary targets, the oligonucleotides are conjugated with red-green fluorescence resonance energy transfer (FRET) donor/quencher pairs.
The authors first confirmed nanoparticle stability and signal generation properties before treatment within MDA-MB-231 cells revealed dual-oligonucleotide release due to dual-fluorophore signal generation. Interestingly, the triplex reportedly exhibits stronger uptake than double helix uptake (99.8% vs. 38.3%). When the triplex conjugates with dendrimers, they utilize micropinocytosis for cellular uptake, triggered by actin-mediated membrane ruffling. By measuring gene activity with PCR and luciferase reporters, the triplex decreases miR-221 expression (oncogenic) and enhances miR-205 expression (tumor suppressor). The triplex is able to suppress cell viability (~90% reduction) and reduce migration ability and colony-forming ability.
To examine the RNA triplex, its pharmacokinetics and therapeutic efficacy are next examined in an orthotopic breast cancer mouse model and compared with doxorubicin (DOX), paclitaxel (PTX), and Avastin monoclonal antibody chemotherapy. Hydrogels loaded with the therapeutic RNA triplex are implanted adjacent to the tumor in the mammary fat pad. While the tumor is tracked with luciferase expression, the release of each oligonucleotide component is monitored with fluorescence over 2 weeks. Bioluminescence imaging reveals that only the RNA triplex containing scaffolds (carrying both miR-205 mimic and antagomiR-221) promotes efficient and sustained inhibition of the tumors with a 90% size reduction. On the other hand, Avastin, hydrogel only, DOX, and PTX loaded hydrogel groups had minimal or no perceptible effect on TNBC tumors.
In fact, tracking the in vivo release of the therapeutic miRNAs suggests a staggered release, indicating that the tumor suppressor pathway is enhanced before the antagomiR silences the activity of tumor-promoting miRNAs. In situ tumor analysis also show that vascularization and cell proliferation are reduced following RNA triplex therapy. Gene expression analysis also corroborates therapeutic efficacy, showing inhibited oncogenes and enhanced immunosuppression by miR-221 target gene downregulation and enhancing miR-205, as well as gene expression of its targets. Thus, Conde and colleagues have developed a versatile and stable RNA triplex–biomaterial therapeutic strategy and demonstrated its efficacy against triple-negative breast cancer (Conde, J.; et al. Nat. Mater.
A Multifunctional Poly(Curcumin) Nanomedicine for Dual-Modal Targeted Delivery, Intracellular Responsive Release, Dual-Drug Treatment, and Imaging of Multi-Drug-Resistant Cancer Cells
In multi-drug-resistant (MDR) cancer cells, intracellular drug accumulation is reduced via excess cellular efflux pumps (e.g., P-glycoprotein [P-gp]). As a means to reduce efflux pump activity, this report presents a novel strategy involving coadministration of pump inhibitors with chemotherapy drugs.
Curcumin, a natural diphenol compound, can suppress P-gp expression and thus sensitize MDR cancer cells and increase chemotherapeutic efficacy. To ensure sufficient bioavailability and therapeutic efficacy, curcumin is incorporated into PEGylated amphiphilic diblock copolymers to improve stability and water solubility. The mixture efficiently self-assembles into stable core/shell nanoparticles, as well as chemotherapeutic drugs. Within the cellular interior, high glutathione (GSH) concentration leads to cleavage of the particle, which leads to curcumin and paclitaxel (PTX) release. To control particle biodistribution and track its whereabouts, magnetic iron oxide and quantum dots are coloaded within the particles.
Initial characterization reveals successful encapsulation of each component, with the nanoparticles (~180 nm) displaying particle stability; magnetic, fluorescent properties; and no agglomeration following the removal of a magnetic field. The polymeric particles have an encapsulation efficiency surpassing 80% of PTX, and minimal PTX release within the first 12 h (<20%). To model nanoparticle behavior in physiological and intracellular situations, 10 mM and 10 µM GSH representing high (intracellular) and low (blood) conditions are modeled. In high GSH conditions (1–10 mM), the particles abruptly release >3-fold (65%) of its PTX cargo, whereas low GSH has no significant effect on release rate.
Using in vitro studies, Wang et al. show that biotin and magnetic targeting improve MCF-7 cell uptake, which they verify with fluorescent imaging of the encapsulated quantum dots. Magnetic targeting further doubled the mean quantity of uptaken nanoparticles with time. After 48 h, a twofold increase in mean fluorescence is observed. By performing cell cytotoxicity assays with increased encapsulated PTX, the nanoparticle with full targeting capability (drug resistance sensitization; magnetic, biotin, controlled PTX release; etc.) decreases IC50 values by >10-fold compared with the free drug and free drug combined with curcumin.
In conclusion, the authors demonstrate a nanoparticle formulation with multiple targeting and bioimaging features that is responsive to tumor cellular environments. Through tumor cell sensitization and targeting moieties, the nanoparticles efficiently reduce drug-resistant cancer cell viability (Wang, J.; et al. J. Mater. Chem. B.
Micro-Nanoscale Automation
Micro- and Nanoscale Technologies for Delivery into Adherent Cells
Currently, there is a dearth of cell-specific, nondestructive tools with suitable temporal or spatial resolution to delineate biological mechanistic complexity. Thus, there is a hindrance to studying cell behavior such as cell heterogeneity, single-cell behavior in complex environments, diseased cells, cell response to stimuli, and stem cell differentiation processes.
In this report, micro- and nanotools for intracellular delivery (in vitro adherent cells) are reviewed. The advantages and limitations of these techniques with respect to cell selectivity, spatial resolution, nondestructive analysis, and high-throughput automation potentially shape future single-cell biological studies.
Traditional intracellular delivery into adherent cells occurs by viral/chemical methods, although these can be cytotoxic or heterogeneous in their results. Micro- and nanotechnology approaches offer better spatiotemporal control and potentially reduce cell stress. Broadly speaking, methods of intracellular delivery can be categorized into single-cell delivery or population-wide cell delivery. Single-cell delivery methods can be further divided into nonfluidic or fluidic nanoprobes. The nonfluidic nanoprobes bear many similarities to conventional microinjection, as the nanostructured tip is controlled with a micromanipulator and monitored relative to the target cell using optical imaging and/or atomic force microscopy (AFM) to quantify cell mechanical properties. Fluidic nanoprobes increase the throughput of cell injection by active control of fluid delivery compared with passive desorption in nonfluidic methods. Fluidic nanoprobes can also incorporate electrochemical measurements, scanning ion channel microscopy, and AFM. Single-dimension (1D) nanostructured arrays have also increased in popularity, although there have been deleterious reports concerning slow growth, abnormal division, DNA damage, and so forth.
Electroporation or electric-assisted intracellular delivery has also been very popular due to its versatility, reproducibility, and ease of usage. The application of a voltage generates transient, reversible pores to facilitate intracellular entry. Variations on this technology include methods that apply electric fields to subcellular regions, mechanical forces, and larger electric fields spanning multiple cell dimensions.
Lab-on-a-chip technologies are versatile platforms that facilitate the combination of microwell arrays with electrical and fluidic controls for automated, multiplexed, and high-throughput delivery of microfluidic quantities. They benefit from having built-in electric circuits to incorporate electrical feedback signals, in addition to optical imaging. Some examples, such as 250 nm diameter nanostraws that combine electroporation, increase plasmid delivery efficiency by >8-fold for CHO cells.
The authors conclude that the development of novel intracellular delivery methods can lead to the development of automated, hands-free cell manipulation and analysis systems. These micro- and nanodevices may even integrate functions such as cell sorting, long-term culture, transfection, sampling, and biomolecule detection with single-cell specificity and high throughput. With improvements in the ability to effectively study single cells, stochastic decision phenomena such as cell differentiation can be uncovered (Kang, W.; et al. Trends Biotechnol.