Photoacoustic spectroscopy of β-hematin

Abstract

Malaria affects over 200 million individuals annually, resulting in 800,000 fatalities. Current tests use blood smears and can only detect the disease when 0.1–1% of blood cells are infected. We are investigating the use of photoacoustic flowmetry to sense as few as one infected cell among 10 million or more normal blood cells, thus diagnosing infection before patients become symptomatic. Photoacoustic flowmetry is similar to conventional flow cytometry, except that rare cells are targeted by nanosecond laser pulses to induce ultrasonic responses. This system has been used to detect single melanoma cells in 10 ml of blood. Our objective is to apply photoacoustic flowmetry to detection of the malaria pigment hemozoin, which is a byproduct of parasite-digested hemoglobin in the blood. However, hemozoin is difficult to purify in quantities greater than a milligram, so a synthetic analog, known as β-hematin was derived from porcine haemin. The specific purpose of this study is to establish the efficacy of using β-hematin, rather than hemozoin, for photoacoustic measurements. We characterized β-hematin using UV-vis spectroscopy, TEM, and FTIR, then tested the effects of laser irradiation on the synthetic product. We finally determined its absorption spectrum using photoacoustic excitation. UV-vis spectroscopy verified that β-hematin was distinctly different from its precursor. TEM analysis confirmed its previously established nanorod shape, and comparison of the FTIR results with published spectroscopy data showed that our product had the distinctive absorbance peaks at 1661 and 1206 cm−1. Also, our research indicated that prolonged irradiation dramatically alters the physical and optical properties of the β-hematin, resulting in increased absorption at shorter wavelengths. Nevertheless, the photoacoustic absorption spectrum mimicked that generated by UV-vis spectroscopy, which confirms the accuracy of the photoacoustic method and strongly suggests that photoacoustic flowmetry may be used as a tool for diagnosis of malaria infection.

Hemozoin-generated vapor nanobubbles for transdermal reagent- and needle-free detection of malaria

“…Such a combination failed to confine the laser-induced thermal effects to iRBCs and thus was not malaria-specific or safe. Previous in vitro photoacoustic approaches (26,27) required a relatively large amount of blood because they used the bulk thermoelastic mechanism of signal generation and thus were unable to detect single iRBCs among many uninfected RBCs. Several other diagnostic methods use the various optical properties of hemozoin (19,(28)(29)(30)(31)(32)(33).…”

“…Hemozoin can be found in any parasite type and any blood stage, including gametocytes (8,10,11). High parasite sensitivity and the specificity of H-VNBs result from a much stronger acoustic signal of a vapor nanobubble compared with that from a bulk thermal effect used by traditional photoacoustics (26,27,34) or from the optical effects in hemozoin (28)(29)(30)(31)(32)(33). Vapor bubbles can also be generated via optical absorbance of hemoglobin in uninfected RBCs but at more than 100-fold higher laser fluence (35).…”

Evanescent Field Based Photoacoustics: Optical Property Evaluation at Surfaces

“…Secondly, EFPA can estimate these properties within a few minutes without complex interaction such as in ellipsometry where input parameter error can cause differences in results. Finally, EFPA, when compared to the traditional photoacoustic effect, has axial resolution 100X smaller as it can estimate thickness of 200 nm films 6 whereas the traditional photoacoustic effect is limited to 7,500 nanometers when using similar lasers and setup 82 . Conveniently, almost every material has independent and differing surface and bulk refractive index properties as well as absorptive properties, which collectively make for functional differences in their use in optical research systems.…”

Rapid transdermal bloodless and reagent-free malaria detection

“…Such a combination failed to confine the laser-induced thermal effects to iRBCs and thus was not malaria-specific or safe. Previous in vitro photoacoustic approaches 28,29 required relatively large amount of blood because they employed the bulk thermoelastic mechanism of signal generation and thus were unable to detect single iRBCs among many uninfected RBCs. Several other diagnostic methods use the various optical properties of hemozoin [30][31][32][33][34][35][36] .…”

“…Hemozoin can be found in any parasite type and any blood stage including gametocytes 8,10,11 . High parasite sensitivity and the specificity of H-VNBs result from a much stronger acoustic signal of a vapor nanobubble compared to that from a bulk thermal effect employed by traditional photoacoustics 28,29,37 or from the optical effects in hemozoin [31][32][33][34][35][36] . Vapor bubbles can also be optically generated via optical absorbance of hemoglobin in uninfected RBCs but at more than 100-fold higher laser fluence 38 .…”