Name: a rapid and chemical-free hemoglobin assay with photothermal angular light scattering
Uploaded: 2016-12-07T15:00:00
Description: Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

Experiments with blood samples were performed in compliance with the relevant laws and institutional guidelines. The samples were the residual blood samples that had been acquired and processed in clinical tests at the institution.
1. PT-AS Optical Setup
NOTE: One may use an empty micro-capillary tube for an initial PT-AS setup.
<ol>
	<li>Mount an empty micro-capillary tube with inner and outer diameters of 200 and 330 &#956;m, respectively, and a length of greater than ~5 cm on a capillary tube fixture. ...

<ol>
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Bioelectron. 57, 59-64 (2014).</li><li>Kim, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary-scale+direct+measurement+of+hemoglobin+concentration+of+erythrocytes+using+photothermal+angular+light+scattering.">Capillary-scale direct measurement of hemoglobin concentration of erythrocytes using photothermal angular light scattering.</a> Biosens. Bioelectron. 74, 469-475 (2015).</li><li>S&#0248;rensen, H. S., Larsen, N. B., Latham, J. C., Bornhop, D. J., Andersen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+biosensing+based+on+interference+from+light+scattering+in+capillary+tubes.">Highly sensitive biosensing based on interference from light scattering in capillary tubes.</a> Appl. Phys. Lett. 89 (15), 151108 (2006).</li><li>Swinney, K., Markov, D., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+volume+refractive+index+detection+using+microinterferometry.">Ultrasmall volume refractive index detection using microinterferometry.</a> Rev. Sci. Instrum. 71 (7), 2684-2692 (2000).</li><li>Tarigan, H. J., Neill, P., Kenmore, C. K., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary-scale+refractive+index+detection+by+interferometric+backscatter.">Capillary-scale refractive index detection by interferometric backscatter.</a> Anal. Chem. 68 (10), 1762-1770 (1996).</li><li>Bornhop, D. 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The PT-AS sensor represents an all-optical method capable of direct [Hb] measurement of unprocessed blood samples. The method quantifies [Hb] in blood using the intrinsic PT response of hemoglobin molecules in erythrocytes. Under illumination by 532-nm light, Hb molecules absorb the light energy and produce heat. The resultant temperature rise changes the RI of the blood sample. The high RI sensitivity of BSI was exploited to measure this RI change in blood. Previously, we demonstrated that the PT-AS sensor enables [Hb] ...

A blood test is commonly performed to evaluate overall human health and to detect biomarkers related to certain diseases. For example, the cholesterol concentration in blood serves as a criterion for hyperlipidemia, which is closely related to cardiovascular diseases and pancreatitis. The blood glucose contents should be measured frequently, as the glucose level is associated with complications such as diabetic ketoacidosis and hyperglycemic hyperosmolar syndrome. Serious illnesses such as malaria, human immunodeficiency virus and acquired immune deficiency syndrome are diagnosed by blood examinations, and quantification of blood components including erythrocytes, thrombocytes, and leukocytes enables screening of pancreatic and renal diseases.
Hemoglobin (Hb), a critical component of blood, makes up about 96% of erythrocytes, and transports oxygen to human organs. Significant alteration of its mass concentration ([Hb]) may indicate metabolic changes, hepatobiliary disease, and neurological, cardiovascular and endocrinological disorders1. [Hb] is therefore routinely measured in blood tests. In particular, anemic patients, dialysis patients, and pregnant women are strongly recommended to monitor [Hb] as a vital task2.
Various [Hb] detection methods have thus been developed. The hemoglobin cyanide method, one of the most common techniques for [Hb] quantification, employs potassium cyanide (KCN) to destroy the lipid bilayer of erythrocytes3. The cyanide hemoglobin produced by the chemical exhibits high absorption around 540 nm; hence, [Hb] measurements can be made via colorimetric analysis. This method is widely employed owing to its simplicity, but the employed chemicals (e.g., KCN and dimethyllaurylamine oxide) are toxic to humans and the environment. The hematocrit scheme measures the volume ratio of red blood cells compared to the total blood volume through centrifugal separation; however it requires a relatively large blood volume (50-100 &#956;l)4. Spectrophotometry methods measure [Hb] precisely without any chemicals, but measurements at multiple wavelengths and a large blood volume are required5,6. Similarly, several optical methods for measuring [Hb] have been proposed including detection methods based on light-scattering, but their measurement accuracies depend strongly on the accuracy of the theoretical blood model.
To overcome these limitations, [Hb] detection methods based on the photothermal (PT) effect of Hb have recently been proposed7. Hb, which is composed mainly of iron oxides, absorbs light at 532 nm and converts the light energy into heat8-10. This PT temperature increase can be detected optically by measuring a change in the refractive index (RI) of blood samples. Yim et al. employed spectral-domain optical coherence reflectometry to measure the PT optical path-length change in a blood-containing chamber11. Although the method enables chemical-free and direct [Hb] measurement, the use of a spectrometer and an interferometric arrangement may hinder its miniaturization. We recently presented an alternative [Hb] detection method, termed photo-thermal angular light scattering (PT-AS) sensor, which is more suitable for device miniaturization12. The PT-AS sensor exploits the high RI sensitivity of the back-scattering interferometry (BSI) to measure PT changes in the RI of a blood sample inside a capillary tube. BSI have been utilized to measure RI of various solutions13-15 and to monitor biochemical interactions in free solution16. The PT-AS sensor employs similar optical arrangement as in BSI, but combines photothermal excitation setup to measure PT increase of RI in blood samples. Operating principles of the BSI and the PT-AS sensors are described in detail elsewhere12,15. PT-AS sensor demonstrated high-sensitivity [Hb] measurement over a wide detection range (0.35-17.9 g/dl) and is capable of operating with sample volumes of &#60;100 nl. No preconditioning of blood sample is required, and the measurement time is only ~5 sec. Here, the experimental setup and a detailed measurement protocol are described. Representative PT-AS results are provided using blood samples from anemic patients, and the results are compared against those from a hematology analyzer to assess the accuracy of the PT-AS sensor.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>650nm laser pointer</td>
			<td>LASMAC</td>
			<td>LED-1</td>
			<td>Probe light</td>
		</tr>
		<tr>
			<td>Hollow round glass capillaries</td>
			<td>Cm Scientific</td>
			<td>CV2033</td>
			<td>Blood sample container</td>
		</tr>
		<tr>
			<td>Webcam</td>
			<td>Logitech</td>
			<td>C525</td>
			<td>CMOS optical sensor</td>
		</tr>
		<tr>
			<td>532-nm DPSS laser</td>
			<td>CNI Laser</td>
			<td>MGL-&#8546;-532</td>
			<td>Photothermal light source</td>
		</tr>
		<tr>
			<td>Optical chopper system</td>
			<td>Thorlabs</td>
			<td>MC2000-EC</td>
			<td>Optical chopper</td>
		</tr>
		<tr>
			<td>Plastic long-pass filter</td>
			<td>Edmund Optics</td>
			<td>#43-942</td>
			<td>To reject 532-nm PT excitation light</td>
		</tr>
		<tr>
			<td>Fiber clamp</td>
			<td>Thorlabs</td>
			<td>SM1F1-250</td>
			<td>Capillary tube fixture</td>
		</tr>
		<tr>
			<td>EDTA coated blood sampling tube</td>
			<td>Greiner Bio-One</td>
			<td>VACUETTE 454217</td>
			<td>Blood sampling &#38; anticoagulating</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Siemens AG</td>
			<td>ADVIA 2120i</td>
			<td>Reference hematology analyzer</td>
		</tr>
	</tbody>
</table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A hemoglobin assay was performed using the PT-AS sensor, and its measurements were compared with those from a hematology analyzer. The experiment was conducted with a PT excitation light intensity of 1.4 W/cm2, PT modulation frequency of 2 Hz, and measurement time of 5 sec. Table 1 summarizes the experimental conditions. The beam sizes of the probe and PT excitation light were 5.5 and 2 mm, respectively. The webcam recorded the images at a frame rate of 30 fps....

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

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

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

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.

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