An Excel fitting routine for correcting protein absorption spectra for scatter.

Protein concentrations are often determined in a non-destructive manner by measuring the absorbance at 280 nm. However, light scattering in protein samples can complicate such assessment. We here describe a simple Excel Solver-based fitting routine to correct full protein UV absorption spectra for both Rayleigh and Mie scattering. Using samples displaying various degrees of natural and artificially induced scattering, we show that our multi-wavelength fitting method is not only capable of aiding in the determination of protein concentrations but can also be employed in the spectral analysis of protein structural changes that are accompanied by alterations in scatter intensity.

Emulsion-based isothermal nucleic acid amplification for rapid SARS-CoV-2 detection via angle-dependent light scatter analysis.

The SARS-CoV-2 pandemic, an ongoing global health crisis, has revealed the need for new technologies that integrate the sensitivity and specificity of RT-PCR tests with a faster time-to-detection. Here, an emulsion loop-mediated isothermal amplification (eLAMP) platform was developed to allow for the compartmentalization of LAMP reactions, leading to faster changes in emulsion characteristics, and thus lowering time-to-detection. Within these droplets, ongoing LAMP reactions lead to adsorption of amplicons to the water-oil interface, causing a decrease in interfacial tension, resulting in smaller emulsion diameters. Changes in emulsion diameter allow for the monitoring of the reaction by use of angle-dependent light scatter (based off Mie scatter theory). Mie scatter simulations confirmed that light scatter intensity is diameter-dependent and smaller colloids have lower intensity values compared to larger colloids. Via spectrophotometers and fiber optic cables placed at 30° and 60°, light scatter intensity was monitored. Scatter intensities collected at 5 min, 30° could statistically differentiate 10, 103, and 105 copies/µL initial concentrations compared to NTC. Similarly, 5 min scatter intensities collected at 60° could statistically differentiate 105 copies/µL initial concentrations in comparison to NTC. The use of both angles during the eLAMP assay allows for distinction between high and low initial target concentrations. The efficacy of a smartphone-based platform was also tested and had a similar limit of detection and assay time of less than 10 min. Furthermore, fluorescence-labeled primers were used to validate target nucleic acid amplification. Compared to existing LAMP assays for SARS-CoV-2 detection, these times-to-detections are very rapid.

Angular photodiode array-based device to detect bacterial pathogens in a wound model.

We have developed a device that is able to rapidly and specifically diagnose bacterial pathogens in a wound model based on Mie scatter spectra from a tissue surface. The Mie scatter spectra collected is defined as the intensity of Mie scatter over the angle of detection from a tissue surface. A 650 nm LED perpendicular to the surface illuminates a tissue sample (90°) and photodiodes positioned in 10° increments from 10° to 80° of backscatter act as the detectors to collect these Mie scatter spectra. Through principal component analysis of the Mie scatter spectra collected, we have shown significant differences between Mie scatter spectra of tissues with bacterial pathogens versus those without, as well as significant differences between each species of bacteria tested. The device developed has been tested with a porcine dermis wound model, with samples inoculated with one of three bacterial species (Staphylococcus aureus, Escherichia coli, or Salmonella Typhimurium). Such a device could be critical in the monitoring of a wound for infection and rapid, specific diagnosis of a bacterial wound infection, which would significantly reduce the time and cost associated with specific diagnosis of a bacterial wound infection currently.

Mie Scatter and Interfacial Tension Based Real-Time Quantification of Colloidal Emulsion Nucleic Acid Amplification.

This work demonstrates for the first time rapid, real-time Mie scatter sensing of colloidal emulsion nucleic acid amplification directly from emulsion droplets. Loop-mediated isothermal amplification is used in this study, and, to our knowledge, has not previously been used in a colloidal emulsion platform. Interfacial tension values (γ) associated with bulk protein adsorption and denaturation at the oil-water interface exhibit characteristic changes in the absence or presence of amplification. In the presence of target and amplicon, emulsions maintain a constant 300-400 nm diameter, whereas emulsions formed with no target control show a rapid decrease in droplet diameter to <100 nm over the first 20 min of incubation. This method is validated using whole bacteria (Staphylococcus aureus MSSA and Escherichia coli O157:H7) and whole virus (Potato virus Y and Zika virus) samples suspended in water, buffer, or serum-like matrices. Short-term formation of colloidal emulsion is quantified via 60° scatter monitoring, where the initial slope of scattering intensity is utilized to confirm target amplification in less than 5 min. The unique benefits of this method render it more cost-effective and field-deployable than existing methods, while being adaptable to a multitude of targets, sample matrices, and nucleic acid amplification tests.

Smartphone-based optofluidic lab-on-a-chip for detecting pathogens from blood.

A novel smartphone-based detection device was created to detect infectious pathogens directly from diluted (10%) human whole blood. The model pathogen was histidine-rich protein 2 (HRP-2), an antigen specific to Plasmodium falciparum (malaria). Anti-HRP-2-conjugated submicrobeads were mixed with HRP-2-infused 10% blood in a lab-on-a-chip device. The white LED flash and the digital camera of the smartphone were used as light source and detector, which delivered light to and from the bead and blood mixture via optofluidic channels in the lab-on-a-chip. The optofluidic channels were angled at 45 degrees to capture the Mie scatter from the sample. Considering the absorption and scattering characteristics of blood (red/infrared preferred) and the Mie scatter simulations for microbead immunoagglutination (UV preferred), blue detection showed the best results. The detection limit was 1 pg/mL in 10% blood. The linear range was from 1 pg/mL to 10 ng/mL. A handheld device, easily attachable to a single smartphone, was finally designed and fabricated using optical mirrors and lenses and successfully detected the HRP-2 from 10% blood. The total assay time was approximately 10 min. The proposed device can potentially be used for detecting a wide range of blood infection with high sensitivity.