Design and Analytical Evaluation of a Rapid Plasma Screening Assay for Circulating Human Papillomavirus DNA via Thermostable Enzyme Chemistries.

Infection with oncogenic strains of human papillomavirus (HPV), such as HPV-16 and HPV-18, can lead to malignant progression and tumorigenesis. As an adjunct to traditional invasive tissue sampling methods, the use of modern thermostable enzyme chemistries can aid in the development of innovative assay workflows to extract and detect circulating HPV DNA (cHPV-DNA) in liquid biopsies. In this work, we first successfully generated a model system to replicate fragmented cHPV-DNA in human plasma. Using this model system, we designed a novel thermostable enzyme chemistry-based cHPV-DNA assay for rapid clinical HPV screening and robustly evaluated its analytical assay performance. Our findings demonstrated that the use of thermostable enzymes provided faster cHPV-DNA extraction and amplification, leading to an overall three-fold improvement in overall assay time as compared to the current standard assay workflow and achieving clinically relevant levels of analytical specificity, sensitivity, and precision for accurate cHPV-DNA detection with excellent 100% sensitivity and specificity in contrived human plasma specimens. In summary, we have devised a rapid laboratory workflow to facilitate the emerging use of liquid biopsies for minimally invasive, rapid, and scalable HPV DNA testing. With facile assay modifications, our thermostable enzyme-based cHPV-DNA assay can be utilized for the detection of other clinically high-risk HPV genotypes.

Tetraplex Immunophenotyping of Cell Surface Proteomes via Synthesized Plasmonic Nanotags and Portable Raman Spectroscopy.

Multiplex immunophenotyping of cell surface proteomes is useful for cell characterization as well as providing valuable information on a patient's physiological or pathological state. Current methods for multiplex immunophenotyping of cell surface proteomes still have associated technical pitfalls in terms of limited multiplexing capability, challenging result interpretation, and large equipment footprint limited to use in a laboratory setting. Herein, we presented a portable surface-enhanced Raman spectroscopy (SERS) assay for multiplex cell surface immunophenotyping. We synthesized and functionalized customizable SERS nanotags for cell labeling and subsequent signal measurement using a portable Raman spectrometer. We extensively evaluated and validated the analytical assay performance of the portable SERS immunophenotyping assay in two different cellular models (red blood cells and breast cancer cells). In terms of analytical specificity, the cell surface immunophenotyping of both red blood cells and breast cancer cells correlated well with flow cytometry. The portable SERS immunophenotyping assay also has comparable analytical repeatability to flow cytometry, with coefficient of variation values of 21.89-23.33% and 6.88-17.32% for detecting red blood cells and breast cancer cells, respectively. The analytical detection limits were 77 cells/mL for red blood cells and 1-17 cells/mL for breast cancer cells. As an alternative to flow cytometry, the portable SERS immunophenotyping assay demonstrated excellent analytical assay performance and possessed advantages such as quick sample-to-result turnaround time, multiplexing capability, and small equipment footprint.

Nucleic Acid Hybridization-Based Noise Suppression for Ultraselective Multiplexed Amplification of Mutant Variants.

The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific "noise" signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed "locker probes" for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized "proximity-programmed" (SERS) readout which allows precise control of hybridization-based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal-free quantification in the Raman silent region (≈1800-2600 cm-1 ). With this dual molecular hybridization-based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.

Separation of distinct exosome subpopulations: isolation and characterization approaches and their associated challenges.

Exosomes are nano-sized extracellular vesicles that serve as a communications system between cells and have shown tremendous promise as liquid biopsy biomarkers in diagnostic, prognostic, and even therapeutic use in different human diseases. Due to the natural heterogeneity of exosomes, there is a need to separate exosomes into distinct biophysical and/or biochemical subpopulations to enable full interrogation of exosome biology and function prior to the possibility of clinical translation. Currently, there exists a multitude of different exosome isolation and characterization approaches which can, in limited capacity, separate exosomes based on biophysical and/or biochemical characteristics. While notable reviews in recent years have reviewed these approaches for bulk exosome sorting, we herein present a comprehensive overview of various conventional technologies and modern microfluidic and nanotechnological advancements towards isolation and characterization of exosome subpopulations. The benefits and limitations of these different technologies to improve their use for distinct exosome subpopulations in clinical practices are also discussed. Furthermore, an overview of the most commonly encountered technical and biological challenges for effective separation of exosome subpopulations is presented.

Native MicroRNA Targets Trigger Self-Assembly of Nanozyme-Patterned Hollowed Nanocuboids with Optimal Interparticle Gaps for Plasmonic-Activated Cancer Detection.

The modernized use of nucleic acid (NA) sequences to drive nanostructure self-assembly has given rise to a new class of designed nanomaterials with controllable plasmonic functionalities for broad surface-enhanced Raman scattering (SERS)-based bioanalysis applications. Herein, dual usage of microRNAs (miRNAs) as both valuable cancer biomarkers and direct self-assembly triggers is identified and capitalized upon for custom-designed plasmonic nanostructures. Through strict NA hybridization of miRNA targets, Au nanospheres selectively self-assemble onto hollowed Au/Ag alloy nanocuboids with ideal interparticle distances (≈2.3 nm) for optimal SERS signaling. The intrinsic material properties of the self-assembled nanostructures further elevate miRNA detection performance via nanozyme catalytic SERS signaling cascades. This enables fM-level miR-107 detection limit within a clinically-relevant range without any molecular target amplification. The miRNA-triggered nanostructure self-assembly approach is further applied in clinical patient samples, and showcases the potential of miR-107 as a non-invasive prostate cancer diagnostic biomarker. The use of miRNA targets to drive nanostructure self-assembly holds great promise as a practical tool for miRNA detection in disease applications.

"Mix-to-Go" Silver Colloidal Strategy for Prostate Cancer Molecular Profiling and Risk Prediction.

Molecular profiling via analysis of multiple disease biomarkers is a powerful tool for disease diagnosis and risk prediction. Due to simplicity and minimal instrumentation requirement, colloidal-based colorimetric DNA/RNA assays are attractive for driving molecular profiling toward widespread clinical usage. Still, the reliability and speed of current colorimetric assays need to be further improved upon for eventual clinical use. Herein, we propose a "mix-to-go" colloidal strategy that utilizes the electrostatic attraction between negatively charged target sequences and positively charged silver nanoparticles (AgNPs) to induce aggregation of AgNPs to profile a panel of clinically validated urinary prostate cancer (PCa) RNA biomarkers ( TMPRSS2:ERG, T2:ERG; prostate cancer antigen 3, PCA3; and kallikrein-related peptidase 2, KLK2). Our strategy is unique in inducing a rapid (10 s), unambiguous solution color change in the presence of target sequences, without the additional NP aggregation agents that are used in existing electrostatic-mediated aggregation assays. Our strategy is analytically specific and sensitive for the detection of 105 and 104 target copies by the naked eye and UV-vis spectrophotometry, respectively. Analytical accuracies of our strategy in detecting T2:ERG, PCA3, and KLK2 RNA biomarkers were 95.9%, 97.3%, and 100%, respectively, as validated by quantitative reverse transcription-polymerase chain reaction. To further evaluate clinical molecular profiling performance beyond conventional proof-of-concept demonstration, we tested our colloidal strategy for noninvasive PCa risk prediction of 73 patients, using the urinary RNA biomarker panel comprising of T2:ERG, PCA3, and KLK2. We found that elevated T2:ERG and PCA3 levels were positively associated with high-risk PCa and obtained a corresponding area-under-the-curve values of 0.790 and 0.833 for predicting PCa and high-risk PCa on biopsy, respectively. We believe our "mix-to-go" strategy may serve as a reliable and accessible Ag colloidal-based molecular profiling approach for clinical applications.

Amplification-Free Multi-RNA-Type Profiling for Cancer Risk Stratification via Alternating Current Electrohydrodynamic Nanomixing.

Simultaneous analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA)-multi-RNA-type profiling-is increasingly crucial in cancer diagnostics. Yet, rapid multi-RNA-type profiling is challenging due to enzymatic amplification reliance and RNA-type-dependent characteristics. Here, a nanodevice is reported to uniquely use alterable alternating current electrohydrodynamic (ac-EHD) forces to enhance probe-target hybridization prior to direct native RNA target detection, without target amplification or surface functionalization. To exemplify clinical applicability, noninvasive screening of next-generation prostate cancer (PCa) RNA biomarkers (of different types) in patient urine samples is performed. A strong correlation between multi-RNA-type expression and aggressive PCa is found, and the nanodevice performance is statistically evaluated. It is believed that this miniaturized system exhibits great potential for cancer risk stratification via multi-RNA-type profiling.

Amplification-Free Detection of Gene Fusions in Prostate Cancer Urinary Samples Using mRNA-Gold Affinity Interactions.

A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing a high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a detectable gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) in urine, noninvasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve the specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models, and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with the current gold standard reverse transcription (RT)-PCR approach with 100% concordance.

Poly(A) Extensions of miRNAs for Amplification-Free Electrochemical Detection on Screen-Printed Gold Electrodes.

Current amplification-based microRNA (miRNA) detection approaches are limited by the small sizes of miRNAs as well as amplification bias/artifacts. Herein, we report on an amplification-free miRNA assay based on elevated affinity interaction between polyadenylated miRNA and bare gold electrode. The poly(A) extension on the 3' ends of magnetically isolated miRNA targets facilitated high adsorption efficiency onto gold electrode surfaces for electrochemical detection without any cumbersome electrode surface functionalization procedures. The assay showed excellent detection sensitivity (10 fM) and specificity and was demonstrated for quantitative miR-107 detection in human cancer cell lines and clinical urine samples. We believe our assay could be useful as an amplification-free alternative for miRNA detection.

Toward Precision Medicine: A Cancer Molecular Subtyping Nano-Strategy for RNA Biomarkers in Tumor and Urine.

Cancer is a heterogeneous disease which manifests as different molecular subtypes due to the complex nature of tumor initiation, progression, and metastasis. The concept of precision medicine aims to exploit this cancer heterogeneity by incorporating diagnostic technology to characterize each cancer patient's molecular subtype for tailored treatments. To characterize cancer molecular subtypes accurately, a suite of multiplexed bioassays have currently been developed to detect multiple oncogenic biomarkers. Despite the reliability of current multiplexed detection techniques, novel strategies are still needed to resolve limitations such as long assay time, complex protocols, and difficulty in interpreting broad overlapping spectral peaks of conventional fluorescence readouts. Herein a rapid (80 min) multiplexed platform strategy for subtyping prostate cancer tumor and urine samples based on their RNA biomarker profiles is presented. This is achieved by combining rapid multiplexed isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) of target RNA biomarkers with surface-enhanced Raman spectroscopy (SERS) nanotags for "one-pot" readout. This is the first translational application of a RT-RPA/SERS-based platform for multiplexed cancer biomarker detection to address a clinical need. With excellent sensitivity of 200 zmol (100 copies) and specificity, we believed that this platform methodology could be a useful tool for rapid multiplexed subtyping of cancers.

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes.

Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide [Fe(CN)6](3-)ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis.

Point-of-need mastitis pathogen biosensing in bovine milk: From academic sample preparation novelty to industry prototype field testing.

Bovine mastitis is an inflammation of the mammary gland, and it is the most common infectious disease in dairy cattle. Mastitis reduces milk yield and quality, costing dairy farmers millions of dollars each year. The aim of this study was to develop a point-of-need test for identifying mastitis pathogens that is field portable, cost-effective and can be used with minimal training. Using a proprietary polymer-based milk sample preparation method to rapidly extract pathogen DNA in milk samples, we demonstrated quantitative Polymerase Chain Reaction (qPCR) assays for six common bovine bacterial mastitis pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Mycoplasma bovis and Escherichia coli. We also implemented this sample preparation method on a prototype point-of-need system in a proof-of-concept field trial to evaluate user experience. Importantly, the protype system enabled a sample-to-result turnaround time of within 70 min to quantitatively detect all six target pathogens. The key advantage of our point-of-need prototype system is being culture-independent yet providing automated milk sample preparation for molecular identification of key mastitis pathogens by non-expert users. Our point-of-need prototype system showed a good correlation to laboratory-based qPCR for target pathogen detection outcomes, thus potentially removing the need for milk samples to be transported off-site for laboratory testing. Above all, we successfully achieved our objective of developing a point-of-need biosensor technology for mastitis and increased its readiness level with industry partners towards technology commercialization.

Nanophotonic immunoarray with electrochemically roughened surfaces for handheld detection of secreted PD-L1 to predict immuno-oncology efficacy.

The analysis of secreted protein biomarkers can be a useful non-invasive method of predicting or monitoring cancer therapeutic response. The increased level of soluble programmed cell death protein ligand 1 (sPD-L1) is a promising predictive biomarker for selecting patients who are likely to respond to immune checkpoint immunotherapy. The current established immunoassay for secreted protein analysis is enzyme-linked immunosorbent assay (ELISA). Yet, ELISA is generally still liable to limited detection sensitivity and restricted to bulky chromogenic readout equipment. Herein, we present a designed nanophotonic immunoarray sensor which achieved sPD-L1 analysis at high-throughput, enhanced detection sensitivity and portability. The key benefits of our nanophotonic immunoarray sensor are (i) high-throughput surface-enhanced Raman scattering (SERS) analysis of multiple samples on a singular platform; (ii) improved sPD-L1 detection sensitivity at 1 pg mL-1 (by two orders of magnitude as compared to ELISA) via electrochemically roughened gold sensor surfaces; (iii) fit for handheld SERS detection with miniaturized equipment footprint. We evaluated the analytical performance of the nanophotonic immunoarray sensor and successfully demonstrated quantitative sPD-L1 detection in a cohort of contrived human plasma samples.

Molecular locker probe enrichment of gene fusion variants from matched patient liquid biopsy specimens for magneto-bioelectrocatalytic nanosensing.

The accurate and sensitive analysis of recurrent gene fusion mutant variants in circulating tumor nucleic acids (NAs) of patient liquid biopsy samples is crucial for realizing clinical potential for cancer screening, diagnostics, and therapeutics. Gene fusion analysis is especially challenging in patient liquid biopsy samples because of trace biotarget levels in high non-target background of highly similar native and variant NA sequences. Herein, we describe accurate analysis of three prostate cancer gene fusion mutant variants in matched plasma and urine specimens from real cancer patients and healthy controls (n = 80) by (i) direct locker probe enrichment of multiple gene fusion mutant variants without tedious upstream sample processing; (ii) magneto-bioelectrocatalytic cycling readout using both NA-intercalating and freely diffusive redox probes for superior signal enhancement. For each mutant variant, an ultrabroad dynamic range (10-105 copies) was achieved with enhanced 10 copies (zmol) detection limit. With the combination of locker probe enrichment and magneto-bioelectrocatalytic cycling readout for NA mutant variant analysis, the potential of non-invasive liquid biopsies may be exploited for the benefit of cancer patients.

Harnessing gene fusion-derived neoantigens for 'cold' breast and prostate tumor immunotherapy.

Breast and prostate cancers are generally considered immunologically 'cold' tumors due to multiple mechanisms rendering them unresponsive to immune checkpoint blockade therapies. With little success in garnering positive outcomes in modern immunotherapeutic clinical trials, it is prudent to re-examine the role of immunogenic neoantigens in these cold tumors. Gene fusions are driver mutations in hormone-driven cancers that can result in alternative mutation-specific neoantigens to promote immunotherapy sensitivity. This review focuses on 1) gene fusion formation mechanisms in neoantigen generation; 2) gene fusion neoantigens in cancer immunotherapeutic strategies and associated clinical trials; and 3) challenges and opportunities in computational and liquid biopsy technologies. This review is anticipated to initiate further research into gene fusion neoantigens of cold tumors for further experimental validation.

Simultaneous BRAFV600E Protein and DNA Aberration Detection in Circulating Melanoma Cells Using an Integrated Multimolecular Sensor.

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.

Bioengineered Polymer Nanobeads for Isolation and Electrochemical Detection of Cancer Biomarkers.

Early sensitive diagnosis of cancer is critical for enhancing treatment success. We previously bioengineered multifunctional core-shell structures composed of a poly-3-hydroxybutyrate (PHB) core densely coated with protein functions for uses in bioseparation and immunodiagnostic applications. Here, we report bioengineering of Escherichia coli to self-assemble PHB inclusions that codisplay a ferritin-derived iron-binding peptide and the protein A-derived antibody-binding Z domain. The iron-binding peptide mediated surface coating with a ferrofluid imparting superparamagnetic properties, while the Z domain remained accessible for binding of cancer biomarker-specific antibodies. We demonstrated that these nanobeads can specifically bind biomarkers in complex mixtures, enabling efficient magnetic separation toward enhanced electrochemical detection of cancer biomarkers such as methylated DNA and exosomes from cancer cells. Our study revealed that superparamagnetic core-shell structures can be derived from biological self-assembly systems for uses in sensitive and specific electrochemical detection of cancer biomarkers, laying the foundation for engineering advanced nanomaterials for diverse diagnostic approaches.

Progressing Antimicrobial Resistance Sensing Technologies across Human, Animal, and Environmental Health Domains.

The spread of antimicrobial resistance (AMR) is a rapidly growing threat to humankind on both regional and global scales. As countries worldwide prepare to embrace a One Health approach to AMR management, which is one that recognizes the interconnectivity between human, animal, and environmental health, increasing attention is being paid to identifying and monitoring key contributing factors and critical control points. Presently, AMR sensing technologies have significantly progressed phenotypic antimicrobial susceptibility testing (AST) and genotypic antimicrobial resistance gene (ARG) detection in human healthcare. For effective AMR management, an evolution of innovative sensing technologies is needed for tackling the unique challenges of interconnected AMR across various and different health domains. This review comprehensively discusses the modern state-of-play for innovative commercial and emerging AMR sensing technologies, including sequencing, microfluidic, and miniaturized point-of-need platforms. With a unique view toward the future of One Health, we also provide our perspectives and outlook on the constantly changing landscape of AMR sensing technologies beyond the human health domain.

Direct Enhanced Detection of Multiple Circulating Tumor DNA Variants in Unprocessed Plasma by Magnetic-Assisted Bioelectrocatalytic Cycling.

The detection of single-nucleotide variants (SNVs) in circulating tumor DNA (ctDNA) in liquid biopsies has increasingly been shown to exhibit unique benefits for early detection or minimal residual disease monitoring in cancer. Yet, current clinically validated assays for ctDNA SNV detection are challenged by (i) time-consuming and laborious spin column-based ctDNA purification protocols, (ii) limited detection specificity to discriminate between mutated SNVs from large excess of closely similar wild-type sequences, and (iii) insufficient detection sensitivity required for trace ctDNA target analysis in blood. Herein, a ctDNA assay is demonstrated to tackle these triple key issues by fusing magnetics for quick ctDNA enrichment directly from unprocessed blood, selected bioenzyme activities for rapid discrimination, and molecular amplification of target SNVs, and designed magnetic-assisted bioelectrocatalytic cycling of DNA-intercalating and freely diffusing redox probes for electrochemical signal intensification. The described ctDNA SNV assay enables the detection of clinically relevant ctDNA SNVs in melanoma (BRAFV600E, KITL576P, and NRASQ61K) from unprocessed plasma samples with unprecedented 0.005% detection sensitivity, ultrabroad dynamic range over four orders of magnitude, and excellent single-base specificity.

Surface-Enhanced Raman Spectroscopy for Cancer Immunotherapy Applications: Opportunities, Challenges, and Current Progress in Nanomaterial Strategies.

Cancer immunotherapy encompasses a variety of approaches which target or use a patient's immune system components to eliminate cancer. Notably, the current use of immune checkpoint inhibitors to target immune checkpoint receptors such as CTLA-4 or PD-1 has led to remarkable treatment responses in a variety of cancers. To predict cancer patients' immunotherapy responses effectively and efficiently, multiplexed immunoassays have been shown to be advantageous in sensing multiple immunomarkers of the tumor microenvironment simultaneously for patient stratification. Surface-enhanced Raman spectroscopy (SERS) is well-regarded for its capabilities in multiplexed bioassays and has been increasingly demonstrated in cancer immunotherapy applications in recent years. This review focuses on SERS-active nanomaterials in the modern literature which have shown promise for enabling cancer patient-tailored immunotherapies, including multiplexed in vitro and in vivo immunomarker sensing and imaging, as well as immunotherapy drug screening and delivery.