Novel Field-Based Protein Detection Method Using Light Transmission Spectroscopy and Antibody Functionalized Gold Nanoparticles.

Protein detection is a universal tool critical to many applications in medicine, agriculture, and biotechnology. We developed a novel protein detection method combining light transmission spectroscopy and particle-size analysis of gold nanospheres monovalently functionalized with polyclonal antibodies and applied it to an emerging challenge for such technologies─the monitoring of environmental proteins (eProteins) present in natural aquatic systems. These are an underreported source of pollution and include the pseudopersistent Cry toxins that enter aquatic ecosystems from surrounding genetically engineered crops. The assay is capable of detecting proteins in complex matrices, such as water samples collected in the field, making it a competitive assay for eProtein detection. It is sensitive, reaching 1.25 ng mL-1, and we demonstrate its application to the detection of Cry1Ab from subsurface tile-drain and streamwater samples from agricultural waterways. The assay can also be quickly adapted for other protein detection applications in the future.

Rapid quantitative protein detection by light transmission spectroscopy.

Rapid, sensitive, and quantitative protein detection is critical for many applications in medicine, environmental monitoring, and the food industry. Advancements in detection of proteins include the use of antigen-antibody binding; however, many current methods are time-consuming and have limiting factors such as low sensitivity and the inability to provide absolute values. We present a new high-throughput method for protein detection using light transmission spectroscopy (LTS), which can quantify and size nanoparticles in fluid suspension. LTS can quantify proteins directly and target specific proteins through antigen-antibody binding. This work shows that LTS can distinguish between and quantify bovine serum albumin, its antibody, and the BSA + Ab complex and determine BSA protein concentrations down to 5 µg/mL. We use both Mie and discrete dipole approximation models to provide geometric insight into the binding process.

Light transmission spectroscopy in real time: a high-resolution nanoparticle analysis instrument.

This paper describes light transmission spectroscopy (LTS), a technique for eliminating spectral noise and systematic effects in real-time spectroscopic measurements. In our work, we combine LTS with spectral inversion for the purpose of nanoparticle analysis. This work employs a wideband multi-wavelength light source and grating spectrometers coupled to CCD detectors. The light source ranges from 210 to 2000 nm, the wavelength-dependent light detection system ranges from 200 to 1100 nm with ≤1 nm resolution, and the nanoparticle diameters range from 1 to 3000 nm. The nanoparticles are suspended in pure water or water-based buffer solutions. For testing and calibration purposes, results are presented for nanoparticles composed of polystyrene and gold. Mie theory is used to model the total extinction cross section, and spectral inversion is employed to obtain quantitative particle size distributions, from which information on the size, shape, and number of nanoparticles can be derived. Discussed are the precision, accuracy, resolution, and sensitivity of our results. The LTS technique is quite versatile and can be applied to spectroscopic investigations where wideband, accurate, low-noise, real-time spectra are desired.

Investigation of signal transduction routes within the sensor/transducer protein BlaR1 of Staphylococcus aureus.

The transmembrane antibiotic sensor/signal transducer protein BlaR1 is part of a cohort of proteins that confer ß-lactam antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) [Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Chem. Rev. 105, 395-424; Llarrull, L. I., Fisher, J. F., and Mobashery, S. (2009) Antimicrob. Agents Chemother. 53, 4051-4063; Llarrull, L. I., Toth, M., Champion, M. M., and Mobashery, S. (2011) J. Biol. Chem. 286, 38148-38158]. Specifically, BlaR1 regulates the inducible expression of ß-lactamases that hydrolytically destroy ß-lactam antibiotics. The resistance phenotype starts with ß-lactam antibiotic acylation of the BlaR1 extracellular domain (BlaRS). The acylation activates the cytoplasmic protease domain through an obscure signal transduction mechanism. Here, we compare protein dynamics of apo versus antibiotic-acylated BlaRS using nuclear magnetic resonance. Our analyses reveal inter-residue interactions that relay acylation-induced perturbations within the antibiotic-binding site to the transmembrane helix regions near the membrane surface. These are the first insights into the process of signal transduction by BlaR1.

Rapid molecular detection of invasive species in ballast and harbor water by integrating environmental DNA and light transmission spectroscopy.

Invasive species introduced via the ballast water of commercial ships cause enormous environmental and economic damage worldwide. Accurate monitoring for these often microscopic and morphologically indistinguishable species is challenging but critical for mitigating damages. We apply eDNA sampling, which involves the filtering and subsequent DNA extraction of microscopic bits of tissue suspended in water, to ballast and harbor water sampled during a commercial ship's 1400 km voyage through the North American Great Lakes. Using a lab-based gel electrophoresis assay and a rapid, field-ready light transmission spectroscopy (LTS) assay, we test for the presence of two invasive species: quagga (Dreissena bugensis) and zebra (D. polymorpha) mussels. Furthermore, we spiked a set of uninfested ballast and harbor samples with zebra mussel tissue to further test each assay's detection capabilities. In unmanipulated samples, zebra mussel was not detected, while quagga mussel was detected in all samples at a rate of 85% for the gel assay and 100% for the LTS assay. In the spiked experimental samples, both assays detected zebra mussel in 94% of spiked samples and 0% of negative controls. Overall, these results demonstrate that eDNA sampling is effective for monitoring ballast-mediated invasions and that LTS has the potential for rapid, field-based detection.

The role of lateral tension in calcium-induced DPPS vesicle rupture.

We assess the role of lateral tension in rupturing anionic dipalmitoylphosphatidyserine (DPPS), neutral dipalmitoylphosphatidylcholine (DPPC), and mixed DPPS-DPPC vesicles. Binding of Ca(2+) is known to have a significant impact on the effective size of DPPS lipids and little effect on the size of DPPC lipids in bilayer structures. In the present work we utilized laser transmission spectroscopy (LTS) to assess the effect of Ca(2+)-induced stress on the stability of the DPPS and DPPC vesicles. The high sensitivity and resolution of LTS has permitted the determination of the size and shape of liposomes in solution. The results indicate a critical size after which DPPS single shell vesicles are no longer stable. Our measurements indicate Ca(2+) promotes bilayer fusion up to a maximum diameter of ca. 320 nm. These observations are consistent with a straightforward free-energy-based model of vesicle rupture involving lateral tension between lipids regulated by the binding of Ca(2+). Our results support a critical role of lateral interactions within lipid bilayers for controlling such processes as the formation of supported bilayer membranes and pore formation in vesicle fusion. Using this free energy model we are able to infer a lower bound for the area dilation modulus for DPPS (252 pN/nm) and demonstrate a substantial free energy increase associated with vesicle rupture.

Quantitative and rapid DNA detection by laser transmission spectroscopy.

Laser transmission spectroscopy (LTS) is a quantitative and rapid in vitro technique for measuring the size, shape, and number of nanoparticles in suspension. Here we report on the application of LTS as a novel detection method for species-specific DNA where the presence of one invasive species was differentiated from a closely related invasive sister species. The method employs carboxylated polystyrene nanoparticles functionalized with short DNA fragments that are complimentary to a specific target DNA sequence. In solution, the DNA strands containing targets bind to the tags resulting in a sizable increase in the nanoparticle diameter, which is rapidly and quantitatively measured using LTS. DNA strands that do not contain the target sequence do not bind and produce no size change of the carboxylated beads. The results show that LTS has the potential to become a quantitative and rapid DNA detection method suitable for many real-world applications.

High-precision sizing of nanoparticles by laser transmission spectroscopy.

We describe the implementation of precision laser transmission spectroscopy for sizing and counting nanoparticles in suspension. Our apparatus incorporates a tunable laser and balanced optical system that measures light transmission over a wide (210-2300 nm) wavelength range with high precision and sensitivity. Spectral inversion is employed to determine both the particle size distribution and absolute particle density. In this paper we discuss results for particles with sizes (diameters) in the range from 5 to 3000 nm. For polystyrene particles 404 to 1025 nm in size, uncertainties of ±0.5% in size and ±4% in density were obtained. For polystyrene particles from 46 to 3000 nm in size, the dynamic range of the system spans densities from ~10(3)/ml to ~10(10)/ml (5 × 10(-8) to 0.5 vol. %), implying a sensitivity 5 orders of magnitude higher than dynamic light scattering.