A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

The goal of this presentation is to demonstrate a direct and rapid hemoglobin assay of anemic blood samples using a photothermal angular light scattering sensor. The PT-AS sensor is a novel, all optical sensor capable of measuring mass concentration of hemoglobin in unprocessed nanoliter volume blood samples without any chemicals. The sensor can be readily implemented with a low cost consumer electronic devices, such as a laser pointer and webcam.

And so, provides ideal platform for point of care testing devices. The sensor can also be utilized to measure various kinds of biological and chemicals species that exhibit strong photothermal response, such as hemoglobin. The blood samples employed in this presentation are the red steel blood samples that had been acquired and processed in the clinical test at the institution.

To begin setting up the photothermal angular light scattering sensor. First mount a clean capillary tube upright on a capillary tube fixture. Then, mount a 650 nanometer probe laser directed at the tube.

Turn on the laser and check the scattering pattern with a sheet of paper. Next, place a camera without a lens on the other side of the capillary tube at an angle of about 25 to 35 degrees relative to the laser beam. Connect the camera to a computer and open the custom camera recording software.

Check that the scattering pattern is in the center of the camera feed. Then, mount a 532 nanometer photothermal excitation laser directed towards the capillary tube. Ensure that the excitation laser light intersects the probe laser at the capillary tube, but does not directly shine into the camera.

Turn off both lasers and remove the capillary tube from the mount. Place a long pass filter in front of the camera so only the 650 nanometer light is detected. Then, mount an optical chopper with a chopper wheel in front of the 532 nanometer laser.

First derive calibration curve for the PT-AS sensor based on reference samples with known mass hemoglobin concentrations. Then, obtain a fresh whole blood sample drawn into a blood sampling tube containing ethylenediaminetetraacetic acid. Fill a clean capillary tube from the sample.

Place the sample filled capillary tube in the mount. Then, turn on the 650 nanometer laser and check that the scattering pattern is centered correctly on the camera. Turn on the 532 nanometer laser at its highest power and turn on the optical chopper control box.

Set the modulation frequency to two hertz and then start the chopper wheel. Using the custom designed software, record the scattering pattern from the camera. Then, turn off the lasers.

Compute the average scattering pattern for each frame of the recording. Take the Fourier transform and calculate the phase at the peak spatial frequency for each scattering pattern. Plot the phase fluctuation over all of the frames as a function of time.

Then, take the Fourier transform in the Time domain to determine the PT-AS signal magnitude at the PT modulation frequency. Convert the PT-AS signal to the hemoglobin mass concentration using the calibration curve. Three different samples were tested with PT-AS and with a hematology analyzer.

Larger phase fluctuations are observed in samples with higher mass hemoglobin concentrations. The PT-AS signals averaged over 11 measurements corresponded to mean mass hemoglobin concentration values of 5.46, 7.23, and 9.85 grams per deciliter, which were similar to the values obtained with the hematology analyzer. Once properly aligned and calibrated, the PT-AS sensor enables high accuracy measurement of mass concentration of hemoglobin in unprocessed blood samples.

A sensor can be built with low cost consumer electronic devices and requires only a blood loaded microcapillary tube for operation. These unique features of the PT-AS sensor will therefore enable it's wide adoption in clinical laboratories and research limited settings.