SIGNAL ENHANCEMENT IN DISPERSE SOLUTIONS FOR THE ANALYSIS OF BIOMEDICAL SAMPLES BY PHOTOTHERMAL SPECTROSCOPY

Photothermal lens spectrometry not only shows high sensitivity of colored heme protein determination, but also provides a change in the sensitivity compared to the theoretical values due to changes in the heat transfer in dispersed media. This can be used for estimating the size of disperse particles exemplified by hemoglobin cyanide, photothermal examination of the state of existence of hemoglobin in highly saline solutions by changes in photothermal properties upon dissociation of hemoglobin tetramers into dimers and monomers. The example of determination of contrast agents (dyes) in blood as the versification of the platform of photoacoustic/photothermal measurement of circulating blood volume is shown.


Introduction
Photothermal spectroscopy develops in many directions, and since the 1980s, it had been used in various analytical applications, which include highly sensitive photometric measurements and versatile detection schemes in liquid chromatography and capillary electrophoresis [1].Recently, much attention is paid to the applications of photothermal spectroscopy in microfluidic systems (chemical microchips or µ-TAS), which begin to play a key role in analytical chemistry [2].However widespread these well-known areas of photothermal applications are, the potentialities of photothermics in analytical chemistry and chemical analysis are even wider.
In this study, we use PTS measurements of protein heme suspensions at high and trace level of constituents in various media (aqueous solutions).Such disperse media are characterized by a multitude of conversion mechanisms, with the main feature of their simultaneous and competing character.Moreover, the existing of particles with constantly changing configuration provides a random constituent of the signal.These features were observed as functions of dynamic (response profile) and statistic (amplitude fluctuation) parameters of PTS signals and disperse-phase parameters.
The synchronization of the measurements is implemented by in-house written software.The PT spectrometer has a linear dynamic range of the signal of four orders of magnitude (the corresponding range of absorption coefficients for 10 mm optical pathways is 1  10 -6 to 2  10 -2 cm -1 ) and response time of 0.005-2 s (depending on the selected measurement parameters, namely, on the data throughput rate and time, the number of points to be averaged, etc.).The spectrometer implements a secondary channel (for gathering scattering signal, if present).The probe beam was reflected by a dichroic mirror; the residual excitation beam was removed with a stained-glass bandpass filter and after a 2-mm pinhole appeared at the primary PT detector.If the photometric or PT channel is not needed, the corresponding detector is switched off.The scattering at the excitation wavelengths was collected with the secondary photodiode near the flowing cell and used to correct the absorbance value, see below.

Flow manifold for in vitro measurements
The flow manifold consisted of a circular flow system pumped with a Watson-Marlow 501U peristaltic pump (UK), a cylindrical flow cell (l = 15 mm; 16 cm 3 ) and a changeable (volumeadjusting) vessel (volume 0.25 to 6 L).
All the tubing parts were from a KDM bloodtransfusion system (KD Medical GmbH, Germany; length 90 cm, i.d.0.3 cm).The system was filled with doubly distilled water, PBS, or stabilized rat blood.The dye was injected through an injected valve before the measurement cell to emulate the injection of the target dye in in vivo tests.The flow rate was kept at 35 ± 1 mL/min (linear velocity 2 cm/s).After each measurement, the working liquid was drained to waste and washed with distilled water through a secondary injection valve.The measurements were made using the PT setup or with a Shimadzu UV Mini 1240 spectrophotometer, Japan (optical absorbance measurements).

Data Treatment
The instrumental signal during an excitation on-off where I(0) and I(t) are intensities of the probe beam in the centre of a detector during the heating part of the chopper cycle.According to the diffraction theory of thermal lensing [4][5][6], the interrelation between the absorption-based (analytical) signal  (see below, (5)) and the instrumental  signal is ) where B(t) is the time-dependent geometryconfiguration constant of thermal-lens spectrometer [7].Pe is the excitation power; A is absorbance; and E0 is the enhancement factor of thermal lensing: Here, p is the probe wavelength; dn/dT is the temperature gradient of the refractive index, and k is the thermal conductivity.
Each determination was characterized by the limit of detection (LOD) calculated as the analyte concentration that produces a thermal lens signal which is three times greater than the standard deviation of the blank solution (3-criterion).The limit of quantification (LOQ) calculated from the RSDrlc = (c) curves is the analyte concentration that produces estimates having a relative standard deviation of 10% (10-criterion) The minimum detectable linear absorption coefficients for PT and photometric (PM) measurements were calculated according to the equations previously deduced from the theory of these two methods for the conditions of shot noise determining the measurement precision [17] Here, h is Planck constant, e and p are frequencies of the excitation and probe beams;  is the detector quantum yield; f is the detection channel bandwidth;  is chopper frequency in PT measurements; B∞ is the steady-state geometry constant of the setup optical scheme, other parameters are listed above.

CBV assessment
We used dyes for intravenous administration for simultaneous PT and optical absorbance determination of components of their two-dye mixtures in circulating blood.CBV were measured as an average of two PT signals for each dye to decrease the interference of both dyes on one another and thus to improve the accuracy (and determined after their dilution in circulation.The concentration of each dye diminishes due to dilution.From the curve of the relative decrease in the concentration, CBV is calculated from the ratio of concentrations of the initial dye solution and the solution of dye in the blood according to the following equation [18][19][20][21]: where  is PT signal amplitude, A is absorbance, V0 and c0 are initial volume and concentration of the dye solution, and cx is the dye concentration in the blood after the dilution.The determination of two components a and b of a dual-component mixture was made using two methods (i) a standard Vierordt's method at two wavelengths λ1 and λ2 [22][23][24]: and (ii) using an overdetermined Vierordt's system at four wavelengths to decrease the overall error [22]:

Here
A is absorbance acquired from spectrophotometric measurements or calculated from PT measurements.The maxima of functions () were used as the wavelengths for Vierordt's method.For the overdetermined Vierordt's system (11), λ1 and λ3 were at the maxima, and λ2 and λ4at the minima of the absorption spectra of a and b components.The calculations of dye concentration were made taking into account the condition сa/сb = const, which is correct for pre-prepared twodye mixtures injected into the flow [22].
The blood of rats stabilized with heparin was used at the stages of blood batch and flow tests.

Determination of label dyes under batch conditions
Stock solutions of selected dyes in blood (0.40 mL of the stock solution of the corresponding dye and 0.60 mL of blood) were used.This freshly prepared stock solution was diluted in the photometric cells by adding a 0.01 -0.05 mL of this solution to 0.40 mL of blood.The calibrations were made at wavelengths 615, 630, 663, and 690 nm, and the limits of detection and other performance parameters were measured.

In vitro assessment of circulating blood volume
The main glass vessel of the manifold was filled with the precisely measured blood volume, and the blood started circulating through the manifold.When the regular flow through the cell is established, the zero absorbance is calibrated.Next, 0.4 mL of an aliquot of stock solutions of MB and CV or their mixture of was injected, and the absorbances at 615, 630, 663, and 690 nm was recorded until constant absorbance/PT value are reached.The concentrations of labels were determined from Eqs. (11).

Back-synchronized spectrometer
We developed a PT-lens flow spectrometer working in batch and flow conditions using the available flow module and an absorption spectroscopy unit to realtime measure of absorption spectra of dye alone and in mixtures.In this excitation/data-treatment mode [3,25], the advantages are (i) the possibility of detection under batch and flow conditions with no change in the optical-scheme design of the instrument; (ii) the possibility to switch between transient and steady-state thermal-lens measurements within a single set of experiments; and (iii) a wide linear dynamic range (more than five orders of magnitude of detectable absorbances) including strongly absorbing and scattering samples.In spite of a somewhat lower detection sensitivity compared to more commonly used lock-in detection schemes in thermal-lens spectrometry, the flexibility of this measurement mode provide a much larger volume of information, making it a powerful tool for solving so not-straightforward problems like studies of complex formation at trace concentrations, timeresolved heat dynamics around absorbing nanoparticles, etc. [22,[26][27][28][29][30].
The setup (Fig. 1) was made using the previously optimized schematics taking into account the precision of measurements and the linear range of thermal lens signal.The main idea is to combine photometric and PT measurements in a single setup.This idea was supported by our previous data on the use of the thermal-lens spectrometer as a singlewavelength photometer providing enough measurement precision [3].
The probe wavelength of 808 nm (marked grey in Fig. 1) was selected as it provided the minimum light absorption of the blood components according to our previous findings [31] and selected dyes and very low scattering.The probe intensity was attenuated to 1 mW at the output and ca.0.7 mW at the cell.The excitation beam was made of four beams of diode lasers with the same beam parameters and the wavelengths of 610, 635, 660, and 690 nm.In a more advanced mode, the diode laser with the same beam parameters and the wavelength of 532 nm was added.The lasers are chopped in sequence synchronized with the main light chopper; thus, every excitation on-off cycle of the thermal lens corresponds to the certain wavelength, and the frequency of the excitation with the same wavelength is /4 (or /5 for a five-wavelength mode).
In order to get the maximum information for a flowing sample (at a flow rate of 35 mL/min), high chopper frequencies are preferable, while the sensitivity of PT measurements decrease with the chopper frequency [12].Thus, a compromise frequency of 10 Hz was used, which corresponded to 2.5 Hz frequencies for a single wavelength.This corresponded to the measurement of ca.0.15 mL of the flowing sample with a certain wavelength and 0.45 mL with other wavelengths, in cycles.
To implement dual-mode measurements, we used a dichroic mirror to separate the probe beam from the excitation.The whole excitation beam after the cell penetrated the dichroic mirror and was gathered with a focusing lens by the primary photometric detector, which was compared with pre-calibrated power meter used as the absorbance ground signal.The selection of the optimum parameters of PT measurements are discussed previously [3,25].

Heme protein studies
The PT determination of some Hb species (desoxyhaemoglobin and oxyhaemoglobin shows the limits of detection at the level of 10 -8 M as in the previous study [31] but with lower errors due to the use of multiple wavelengths.
The experiments with haemoglobin cyanide species with the back-synchronized measurement mode showed that the changes in amplitude and timeresolved thermal-lens signal can be used for determining the dispersity of the solution.
The determination of HbCN and ferroin selected as a highly absorbing chelate shows low and silmilar LODs under the excitation conditions (Table 2).However, time-resolved curves for molecular and colloid solutions of HbCN differ (Fig. 2, a) and the characteristic time of thermal-lens measurements, Eq. ( 4), changes significantly (Table 2), while the slope of the calibration plot goes lower than expected.This is in contrast to ferroin, for which the calculated and experimental value are in good agreement.This behavior is due to faster heating of the vicinity of the absorbing disperse particles, which decreases the characteristic time and slower heat transfer into the solution body, which decreases the steady-state thermal lens signal.This model for thermal lensing in HbCN solutions is well simulated by the developed theoretical approach [8,10].
Moreover, the overall change in tc is additive (see Table 2 and Fig. 2, b): an equimolar mixture of HbCN and ferroin shows a expected change in the slope and a shift in the characteristic time.This, also can be predicted by the approach [8,10] however, this requires further development in the theory.
The characteristic time of thermal lens in HbCN solutions depend on the concentration of the protein (Fig. 3).The dependence is described with the empirical equation Here,  , and  , are thermal lens characteristic times for a test disperse system and water, c is disperse particle molarity, d is their average diameter and k1 and k2 are empiric coefficients.From this dependence, the size of hemoglobin cyanide molecule, (6.0 ± 3.0) nm was calculated the developed approach for heterogonous systems in TLS.This value is in good agreement with the size of the hemoglobin globule [32].Moreover, this change in the shape of transient thermal-lens curves makes it possible to estimate the state of these proteins in solution.For hemoglobins, an increase in the ionic strength from (i) standard phosphate buffers to highly saline (ii) NaCl, and (iii) NaClO4 solutions results in the ratio of the TLS signal amplitudes as i : ii : iii of 1 : 2 : 4, which is in good concordance with the existing data [33] for the decomposition of hemoglobin into 2 and 4 subunits, respectively.Measuring the model molecular dye (ferroin) and HbCN under these conditions shows the number of changes.The characteristic times of transient curves and calibration slopes for ferroin do not change as expected (Table 2).To the contrary, the curve for hemoglobin in NaCl start to go much closer to the curve of ferroin (Fig. 4) and tc differ much less significantly (Table 2) due to dissociation of tetramers to dimers; and in NaClO4 solutions the transient curve for HbCN start to differ insignificantly from the ferroin solution due to final dissociation to monomers [33].

PT-lens measurements of the circulating blood volume
The ability of thermal-lensing to change sensitivity in protein solutions was used in the measurement of circulating blood volume (CBV) by the designed setup.CBV is crucial in various medical conditions including surgery, iatrogenic problems, rapid fluid administration, transfusion of red blood cells, or trauma with extensive blood loss including battlefield injuries and other emergency situations loss [18,20,[34][35][36][37]. Currently available commercial techniques are invasive and time-consuming for trauma situations [35,36].Recently, we have proposed high-speed multi-wavelength photoacoustic/photothermal (PA/PT) flow cytometry for in vivo CBV assessment with multiple dyes as PA and PT contrast agents (labels) [22].

Photothermal vs. photometric sensitivity comparison
According to Eq. ( 13), it is possible to compare the sensitivity of photometric and PT measurements and for the same detector and the same laser used for measuring absorption and PT excitation.The comparison of sensitivities for the same detector type ( and f parameters in eq. ( 7)) and the same source of absorption/PT excitation is given by the equation deduced from ( 7) and (8) This equation shows that an increase in the sensitivity depends on the geometry parameters of the setup and the parameters of the medium, in which the thermal lens is bloomed.For the experimental conditions discussed above the calculation shows that the minimum detectable linear absorption coefficient is about 250-fold lower that for photometry.This means that the significant diminishing of dye doses to get reliable PT signals compared to current clinical doses (for PDD, 2.5-5 mg/mL) can be made when shifting from optical photometric to PT measurements of CBV.

Photothermal measurements of model dyes in blood
As additional verification of our proof-of-concept of the PT part of this technique, here we performed optical photometric and PT CBV measurements in vitro with the described above multi-wavelength photothermal-lens spectrometer.Two label dyes-Methylene Blue and Crystal Violet-were selected for simultaneous photometric/PT determination of the components of their two-dye mixtures in the circulating blood in vitro.Working concentrations of both label dyes, MB and CV were diminished by a factor of ca.50 compared to existing CBV methods and to the level used for optical photometric measurements in his study to 10 nmol/l.From absorption spectra, the linear absorption coefficient for MB and CV in blood at the selected wavelengths at the red region are 20-25 cm -1 at 100 µM (0.1 mg/mL) while blood absorption at IR laser line of 808 nm is 4-5 cm -1 at 808 nm [38,39].The examples of flow measurements of both dyes by PT spectrometry are shown in Fig. 5.
The minimum delectable concentrations of MB and CV in blood by PT measurements under the selected conditions are 0.2 and 0.5 µM, respectively which is 250 times lower than the clinically approved doses [39].These values are in good agreement with the theoretical estimations from Eq. discussed in the previous section.The estimations of CBV assessment in the test manifold showed the error of assessment is below 5% for blood volumes 0. compared to photometric measurements.The error of measurements agrees well with our previous data on PA measurements of blood parameters [22,40].

Conclusions
Thermal lens spectrometry can be implemented as a multifunctional device for various fundamental and applied studies on heme proteins and their solutions.This thermal-lens approach can be used for determining state-of-the-art nanomaterials like nanodiamond and fullerenes, which are not considered within the frames of this study.Also, the transient thermal lensing can be used to overcome the diffraction limit of optical photothermal spectroscopy to attain super-resolution of photothermal imaging, which is of high importance for various applications like studies of mitochondria, erythrocyte pathologies and similar problems [41,42].The following studies should be focused on the integration of all the modes of photothermal measurements in a single compact device for micromeasurements [2].

Fig. 2 .Fig. 3 .
Fig. 2. Transient curves of thermal-lens signals from for a molecular dye (ferroin), green line, and hemoglobin cyanide, blue line, with the same absorbance.(a) are full curves for pure substances; (b) are thermal-lens development curves only with the yellow line corresponding to 1 : 1 molar mixture of ferroin and hemoglobin cyanide ),  = 532 nm, excitation power 47.5 mW

Table . 2
. Performance parameters and characteristic times of thermal-lens determination of ferroin as a molecular chelate dye and hemoglobin cyanide under various conditions in a phosphate buffer solution, pH 7.0 and in a highly saline solution (P = 0.95, n = 6),  = 532 nm, excitation power 47.5 mW