Extinction Coefficient of Gold Nanostars

Gold nanostars (NStars) are highly attractive for biological applications due to their surface chemistry, facile synthesis, and optical properties. Here, we synthesize NStars in HEPES buffer at different HEPES/Au ratios, producing NStars of different sizes and shapes and therefore varying optical properties. We measure the extinction coefficient of the synthesized NStars at their maximum surface plasmon resonances (SPRs), which range from 5.7 × 10⁸ to 26.8 × 10⁸ M⁻¹ cm⁻¹. Measured values correlate with those obtained from theoretical models of the NStars using the discrete dipole approximation (DDA), which we use to simulate the extinction spectra of the nanostars. Finally, because NStars are typically used in biological applications, we conjugate DNA and antibodies to the NStars and calculate the footprint of the bound biomolecules.United States. National Institutes of Health (AI100190

Quantifying the nanomachinery of the nanoparticle-biomolecule interface

A study is presented of the nanomechanical phenomena experienced by nanoparticle-conjugated biomolecules. A thermodynamic framework is developed to describe the binding of thrombin-binding aptamer (TBA) to thrombin when the TBA is conjugated to nanorods. Binding results in nanorod aggregation (viz. directed self-assembly), which is detectable by absorption spectroscopy. The analysis introduces the energy of aggregation, separating it into TBA–thrombin recognition and surface-work contributions. Consequently, it is demonstrated that self-assembly is driven by the interplay of surface work and thrombin-TBA recognition. It is shown that the work at the surface is about −10 kJ mol−1 [mol superscript -1] and results from the accumulation of in-plane molecular forces of pN magnitude and with a lifetime of <1 s, which arises from TBA nanoscale rearrangements fuelled by thrombin-directed nanorod aggregation. The obtained surface work can map aggregation regimes as a function of different nanoparticle surface conditions. Also, the thermodynamic treatment can be used to obtain quantitative information on surface effects impacting biomolecules on nanoparticle surfaces.MIT-IQS Exchange Progra

Challenges of the nano-bio interface in lateral flow and dipstick immunoassays

Lateral flow assays (LFAs) are highly attractive for point-of-care (POC) diagnostics for infectious disease, food safety, and many other medical uses. The unique optical, electronic, and chemical properties that arise from the nanostructured and material characteristics of nanoparticles provide an opportunity to increase LFA sensitivity and impart novel capabilities. However, interfacing to nanomaterials in complex biological environments is challenging and can result in undesirable side effects such as non-specific adsorption, protein denaturation, and steric hindrance. These issues are even more acute in LFAs where there are many different types of inorganic–biological interfaces, often of a complex nature. Therefore, the unique properties of nanomaterials for LFAs must be exploited in a way that addresses these interface challenges. LFAs for infectious disease, food safety, and many other applications have been enhanced by nanotechnology. However, interface effects in LFAs are much more complicated, which is problematic because POC devices must be robust, simple, and easy to use. To fully utilize the unique properties of nanotechnology, these interface issues must be understood, controlled, and also leveraged.NIH NIAID (AI100190

Point-of-Care Devices to Detect Zika and Other Emerging Viruses

Rapid diagnostic tests (point-of-care devices) are critical components of informed patient care and public health monitoring (surveillance applications). We propose that among the many rapid diagnostics platforms that have been tested or are in development, lateral flow immunoassays and synthetic biology-based diagnostics (including CRISPR-based diagnostics) represent the best overall options given their ease of use, scalability for manufacturing, sensitivity, and specificity. This review describes the identification of lateral flow immunoassay monoclonal antibody pairs that detect and distinguish between closely related pathogens and that are used in combination with functionalized multicolored nanoparticles and computational methods to deconvolute data. We also highlight the promise of synthetic biology-based diagnostic tests, which use synthetic genetic circuits that activate upon recognition of a pathogen-associated nucleic acid sequence, and discuss how the combined or parallel use of lateral flow immunoassays and synthetic biology tools may represent the future of scalable rapid diagnostics

A comparison of nanoparticle-antibody conjugation strategies in sandwich immunoassays

Point-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers

Programmable CRISPR-responsive smart materials

Stimuli-responsive materials activated by biological signals play an increasingly important role in biotechnology applications. We exploit the programmability of CRISPR-associated nucleases to actuate hydrogels containing DNA as a structural element or as an anchor for pendant groups. After activation by guide RNA–defined inputs, Cas12a cleaves DNA in the gels, thereby converting biological information into changes in material properties. We report four applications: (i) branched poly(ethylene glycol) hydrogels releasing DNA-anchored compounds, (ii) degradable polyacrylamide-DNA hydrogels encapsulating nanoparticles and live cells, (iii) conductive carbon-black–DNA hydrogels acting as degradable electrical fuses, and (iv) a polyacrylamide-DNA hydrogel operating as a fluidic valve with an electrical readout for remote signaling. These materials allow for a range of in vitro applications in tissue engineering, bioelectronics, and diagnostics.Defense Threat Reduction Agency (Grant HDTRA1-14-1-0006

Creating CRISPR-responsive smart materials for diagnostics and programmable cargo release

Materials that sense and respond to biological signals in their environment have a broad range of potential applications in drug delivery, medical devices and diagnostics. Nucleic acids are important biological cues that encode information about organismal identity and clinically relevant phenotypes such as drug resistance. We recently developed a strategy to design nucleic acid–responsive materials using the CRISPR-associated nuclease Cas12a as a user-programmable sensor and material actuator. This approach improves on the sensitivity of current DNA-responsive materials while enabling their rapid repurposing toward new sequence targets. Here, we provide a comprehensive resource for the design, synthesis and actuation of CRISPR-responsive hydrogels. First, we provide guidelines for the synthesis of Cas12a guide RNAs (gRNAs) for in vitro applications. We then outline methods for the synthesis of both polyethylene glycol-DNA (PEG-DNA) and polyacrylamide-DNA (PA-DNA) hydrogels, as well as their controlled degradation using Cas12a for the release of cargos, including small molecules, enzymes, nanoparticles and living cells within hours. Finally, we detail the design and assembly of microfluidic paper-based devices that use Cas12a-sensitive hydrogels to convert DNA inputs into a variety of visual and electronic readouts for use in diagnostics. Following the initial validation of the gRNA and Cas12a components (1 d), the synthesis and testing of either PEG-DNA or PA-DNA hydrogels require 3–4 d of laboratory time. Optional extensions, including the release of primary human cells or the design of the paper-based diagnostic, require an additional 2–3 d each.Defense Threat Reduction Agency (Grant HDTRA1-14-1-0006

Wearable materials with embedded synthetic biology sensors for biomolecule detection

Integrating synthetic biology into wearables could expand opportunities for noninvasive monitoring of physiological status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightweight, flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chemicals and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aqueous exposure events and report the presence of specific molecular targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current laboratory methods such as quantitative PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temperature within 90 min, requiring no user intervention other than the press of a button

Development and validation of a rapid lateral flow E1/E2-antigen test and ELISA in patients infected with emerging Asian strain of Chikungunya virus in the Americas

Since its 2013 emergence in the Americas, Chikungunya virus (CHIKV) has posed a serious threat to public health. Early and accurate diagnosis of the disease, though currently lacking in clinics, is integral to enable timely care and epidemiological response. We developed a dual detection system: a CHIKV antigen E1/E2-based enzyme-linked immunosorbent assay (ELISA) and a lateral flow test using high-affinity anti-CHIKV antibodies. The ELISA was validated with 100 PCR-tested acute Chikungunya fever samples from Honduras. The assay had an overall sensitivity and specificity of 51% and 96.67%, respectively, with accuracy reaching 95.45% sensitivity and 92.03% specificity at a cycle threshold (Ct) cutoff of 22. As the Ct value decreased from 35 to 22, the ELISA sensitivity increased. We then developed and validated two lateral flow tests using independent antibody pairs. The sensitivity and specificity reached 100% for both lateral flow tests using 39 samples from Colombia and Honduras at Ct cutoffs of 20 and 27, respectively. For both lateral flow tests, sensitivity decreased as the Ct increased after 27. Because CHIKV E1/E2 are exposed in the virion surfaces in serum during the acute infection phase, these sensitive and specific assays demonstrate opportunities for early detection of this emerging human pathogen.U.S. Public Health Service (award AI100190

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics