Smartphone-Based Device for Non-Invasive Heart-Rate Measurement of Chicken Embryos.

Heart rate (HR) is an important parameter in the study of the developmental physiology of chicken embryos and a crucial indicator of dead or live embryo grading in artificial incubation processes. A non-invasive HR measurement technique is required for long-term and routine HR assessment with minimal influence on embryo development. Accordingly, in this study, a non-invasive HR measurement technique of chicken embryos using a smartphone is demonstrated. The detection method of the proposed device is based on the photoplethysmography principle in which a smartphone camera is used for video recording, and the chicken embryonic HR is obtained from the recorded video images using a custom Android application. We used a smartphone to measure the embryonic HR of 60 native chicken eggs and found that it can measure the chicken embryonic HR from day 4 to day 20. The proposed smartphone HR device will be beneficial for scientific research and industrial applications. With internet connectivity, users can utilize their smartphone to measure the HR, display, share, and store the results.

Significant Sensitivity Improvement for Camera-Based Lateral Flow Immunoassay Readers.

Recent developments in smartphone-based strip readers have further improved the performances of lateral flow test kits. Most smartphone cameras encode an unaltered and nonlinear power-law transfer function that maps the light intensity to a pixel value; this poses some limitations for camera-based strip readers. For faint-color test lines which are almost as white such as with nitrocellulose pads, the slope of the transfer function is low. Therefore, it is difficult to differentiate between the faint test lines and the white background. We show that by manually setting the camera exposure time-instead of using the automatic settings-to the high-slope region of the transfer function, the reader's sensitivity can be improved. We found that the sensitivity and the limit of detection of the Acidovorax avenae subsp. citrulli (Aac) test kit were enhanced up to 3-fold and 5-fold, respectively, when using the readers at the optimal camera settings, compared to the automatic mode settings. This simple technique can be readily applied to any existing camera-based colorimetric strip reader to significantly improve its performance.

Rh blood phenotyping (D, E, e, C, c) microarrays using multichannel surface plasmon resonance imaging.

The application of Surface Plasmon Resonance Imaging (SPRi) for the detection of transmembrane antigen of the Rhesus (Rh) blood group system is demonstrated. Clinically significant Rh blood group system antigens, including D, C, E, c, and e, can be simultaneously identified via solid phase immobilization assay, which offers significant time savings and assay simplification. Red blood cells (RBCs) flowed through the micro-channel, where a suitable condition for Rh blood group detection was an RBC dilution of 1:10 with a stop-flow condition. Stop flow showed an improvement in specific binding compared to continuous flow. Rh antigens required a longer incubation time to react with the immobilized antibody than A and B antigens due to the difference in antigen type and their location on the RBC. The interaction between the immobilized antibodies and their specific antigenic counterpart on the RBC showed a significant difference in RBC removal behavior using shear flow, measured from the decay of the SPR signal. The strength of the interaction between the immobilized antibody and RBC antigen was determined from the minimum wall shear stress required to start the decay process in the SPR signal. For a given range of immobilized antibody surface densities, the Rh antigen possesses a stronger interaction than A, B, and AB antigens. Identification of 82 samples of ABO and Rh blood groups using SPRi showed good agreement with the standard micro-column agglutination technique. A wider coverage of antigenic recognition for RBC when using the solid phase immobilization assay was demonstrated for the RBC with the antigenic site located on the transmembrane protein of the clinically significant Rh antigen. Given the level of accuracy and precision, the technique showed potential for the detection of the Rh minor blood group system.

Surface plasmon resonance imaging for ABH antigen detection on red blood cells and in saliva: secretor status-related ABO subgroup identification.

Low antigenic expression of ABO subgroup system on red blood cell (RBC) is cause of discrepancy between forward and reverse blood typing in the standard agglutination technique. Neutralization agglutination is employed for verification of the detection of ABH substances in saliva. However, the neutralization technique is complicated, time-consuming and requires expertise. To overcome these drawbacks, surface plasmon resonance (SPR) imaging was developed for ABH antigen detection on RBCs and in saliva. An antibody array was designed to classify the ABO subgroups by anti-A, anti-B, and anti-H antibodies; the array was immobilized on a carboxymethyl-dextran sensor-surface. RBCs and saliva specimens from sixty-four donors were analysed by passing them over the antibody array, where the secretor status and blood group could be simultaneously identified. Consequently, the immobilized antibodies could specifically and quantitatively detect the ABH antigen on RBCs. Using the direct assay, the SPR signal of saliva detection was weaker than that of RBC detection. However, a sandwich assay with a mixture of anti-A, anti-B, and anti-H antibodies could efficiently enhance the signal. The sensor chip provided high specificity (cut-off at 100 to 175 micro refractive index units) and high precision at 0.06%-4.9% CV. The blood group results of the sixty-four donor specimens obtained by SPR agreed with the standard agglutination test with 100% accuracy. SPR could indicate different ABH antigen densities on the RBCs and nearly the same amounts of ABH substances in the saliva of strong and weak subgroups. Finally, we also demonstrated reduced assay time and fewer complications with the SPR imaging platform compared to the neutralization technique.

Hemoculture and Direct Sputum Detection of mecA-Mediated Methicillin-Resistant Staphylococcus aureus by Loop-Mediated Isothermal Amplification in Combination With a Lateral-Flow Dipstick.

This study reports loop-mediated isothermal amplification (LAMP) for rapid detection of methicillin-resistant Staphylococcus aureus from direct clinical specimens. Four primers including outer and inner primers were specifically designed on the two target sequences-femB to identify S. aureus and mecA to identify antibiotic-resistant gene. Reference strains including various species of gram-positive/gram-negative isolates were used to evaluate and optimize LAMP assays. The optimum LAMP condition was found at 63°C within 70 min assay time (include hybridization with FITC probe for 5 min and further 5 min for reading the results on the lateral flow dipstick). The detection limits of LAMP for mecA was 10 pg of total DNA or 100 CFU/ml. The LAMP assays were applied to a total of 155 samples of direct DNA extraction from sputum and hemoculture bottles. The sensitivity of LAMP for mecA detection in sputum and hemoculture bottles was 93.3% (28/30) and 100% (52/52), respectively. In conclusion, LAMP assay is an alternative technique for rapid detection of MRSA infection with a technical simplicity and cost-effective method in a routine diagnostic laboratory.

SPR-DNA array for detection of methicillin-resistant Staphylococcus aureus (MRSA) in combination with loop-mediated isothermal amplification.

In this study, we evaluated surface plasmon resonance imaging (SPR imaging) as a DNA biosensor for the detection of methicillin-resistant Staphylococcus aureus (MRSA) which is one of the most common causes of nosocomial infections. The DNA sample were collected from clinical specimens, including sputum and blood hemoculture were undergone LAMP amplification for 0.18 kbp and 0.23 kbp DNA fragments of femB and mecA genes, respectively. The self-assembled monolayer surface (SAMs) was used for immobilized streptavidin-biotinylated probes on the sensor surface for the detection of LAMP amplicons from MRSA. Both LAMP amplicons were simultaneously hybridized with ssDNA probes immobilized onto a bio-functionalized surface to detect specific targets in the multiplex DNA array platform. In addition, the sensor surface could be regenerated allowing at least five cycles of use with a shortened assay time. The detection limit of LAMP-SPR sensing was 10 copies/µl and LAMP-SPR sensing system showed a good selectivity toward the MRSA.

Development and beam-shape analysis of an integrated fiber-optic confocal probe for high-precision central thickness measurement of small-radius lenses.

This work describes a new design of a fiber-optic confocal probe suitable for measuring the central thicknesses of small-radius optical lenses or similar objects. The proposed confocal probe utilizes an integrated camera that functions as a shape-encoded position-sensing device. The confocal signal for thickness measurement and beam-shape data for off-axis measurement can be simultaneously acquired using the proposed probe. Placing the probe's focal point off-center relative to a sample's vertex produces a non-circular image at the camera's image plane that closely resembles an ellipse for small displacements. We were able to precisely position the confocal probe's focal point relative to the vertex point of a ball lens with a radius of 2.5 mm, with a lateral resolution of 1.2 µm. The reflected beam shape based on partial blocking by an aperture was analyzed and verified experimentally. The proposed confocal probe offers a low-cost, high-precision technique, an alternative to a high-cost three-dimensional surface profiler, for tight quality control of small optical lenses during the manufacturing process.

Evaluation of agglutination strength by a flow-induced cell movement assay based surface plasmon resonance (SPR) technique.

A flow-induced cell movement assay combined with a surface plasmon resonance (SPR) technique was developed to quantify the agglutination strength, derived from the standard tube-agglutination test. Red blood cells (RBCs), based on the ABO blood group system, were specifically captured by anti-A and/or anti-B antibodies immobilized on a sensor surface. The agglutination strength corresponds to the amount of antigen-antibody interactions or the strength of RBC adhesion. Under a shear flow, the adherent RBCs were forced to move out of the region of interest with different average cell velocities (vc) depending upon the adhesion strength and wall shear stress (WSS). That is, a higher adhesion strength (higher agglutination strength) or lower WSS represents a lower vc or vice versa. In this work, the agglutination strength was derived from the vc that was calculated from the time derivative of the relative SPR signal by using a simple model of cell movement response, whose validity was verified. The vc values of different samples were correlated with their agglutination strengths at a given WSS and antibody surface density. The vc decreased as the agglutination strength increased, which can be considered as a linear regression. The coefficient of variation of the calculated vc decreased to 0.1 as vc increased to 30 µm min(-1). The sensitivity of this assay can be controlled by optimizing the antibody surface density or the WSS. This assay has the capability to resolve the antigen density of A1 and B RBCs from that of A1B RBCs.

ABO blood-typing using an antibody array technique based on surface plasmon resonance imaging.

In this study, readily available antibodies that are used in standard agglutination tests were evaluated for their use in ABO blood typing by a surface plasmon resonance imaging (SPR imaging) technique. Five groups of antibodies, including mixed clones of anti-A, anti-B, and anti-AB, and single clones of anti-A and anti-B, were used to construct the five-line detection arrays using a multichannel flow cell in the SPR imager. The red blood cell (RBC) samples were applied to a multichannel flow cell that was orthogonal to the detection line arrays for blood group typing. We found that the blood samples were correctly grouped in less than 12 min by the SPR imaging technique, and the results were consistent with those of the standard agglutination technique for all 60 samples. We found that mixed clones of antibodies provided 33%-68% greater change in the SPR signal than the single-clone antibodies. Applying the SPR imaging technique using readily available antibodies may reduce the costs of the antibodies, shorten the measurement time, and increase the throughput.

Development of surface plasmon resonance imaging for detection of Acidovorax avenae subsp. citrulli (Aac) using specific monoclonal antibody.

An immunosensor based on surface plasmon resonance imaging (SPR imaging) using a specific monoclonal antibody 11E5 (MAb 11E5) was developed for the detection of the seed-borne bacterium Acidovorax avenae subsp. citrulli (Aac), which causes fruit blotch in watermelons and cantaloupes, and compared to the conventional ELISA technique. The 1:40 mixed self-assembled monolayer (mixed SAM) surface was used for the immobilized MAb 11E5 on sensor surface for the detection of Aac. Both whole cells and broken cells of Aac were tested by using direct and sandwich detection assay. The limit of detection (LOD) of Aac using the SPR imaging technique and a direct detection assay was 10(6)cfu/ml and a subsequent amplification of the SPR signal using a polyclonal antibody (PAb) lowered the LOD to 5×10(5) cfu/ml. The LOD for the ELISA technique was 5×10(4) cfu/ml for the detection of Aac, which was slightly better than that for the SPR technique. However, the sensor surface based on SPR imaging offered a major advantage in terms of surface regeneration, allowing at least five cycles with a shorter time assay, multi-channel analysis with an application on multiplex detection, and an ease of the surface usage for the detection of Aac in the naturally infected plant. The surface was tested against the naturally infected sample and showed good selectivity toward the Aac bacteria.