A Vertical Flow Method for Sensitive Raman Protein Measurement in

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A Vertical Flow Method for Sensitive Raman Protein Measurement in Aqueous Solutions Shu-Chi Li† and Hirotsugu Hiramatsu*,†,‡ †

Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, Hsinchu 30010, Taiwan Center for Emergent Functional Matter Science, National Chiao Tung University, Hsinchu 30010, Taiwan

‡

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S Supporting Information *

ABSTRACT: Raman scattering is intrinsically faint. Raman spectroscopy would be more valuable with improvements in the signal detection efficiency. To improve the signal detection efficiency, we propose a vertical flow method, which is a derivative of the liquid core optical fiber technique employed for sensitive Raman signal detection. In the vertical flow method, the sample solution flows from a pinhole to prepare a liquid column wrapped with air and uses total reflection at the sample−air interface to confine the excitation beam and Raman signal. The Raman signal emerges from the pinhole for efficient collection. This method enhanced Raman signal intensity by up to 90 times. When measuring a bovine serum albumin aqueous solution, the limit of detection (LOD) was 0.029 ± 0.003 mg mL−1 (0.44 ± 0.05 μM). We discuss the signal enhancement factor dependence on several parameters in the vertical flow method. Our method provides a simple technique to improve Raman spectroscopy sensitivity using universal materials.

P

method is to increase signal intensity. A sphere glass cell enhances the Raman signal by a factor (hereafter referred to as the signal enhancement factor, SEF) of nearly three, whose results are measured with a capillary glass tube.15 The sphere cell reduced refraction at the sample−glass interface and improved the collection lens acceptance aperture. A microcavity method enhanced the Raman signal of liquid samples, which were placed in an ∼100 μm hemispheric cavity on an Al plate, by several orders of magnitude.16 This enhancement is due to multiple laser excitations, increased sample volume, and a microcavity focusing effect produced with a concave mirror. Previous studies have also proposed the use of long-path cells to increase the signal. The Raman signal can be effectively collected at the end of an optical fiber that consists of the liquid sample as the “core”. The clad confines light due to the total reflection in this fiber, which is referred to as the liquid core optical fiber (LCOF). Walrafen and Stone17 demonstrated the benefits of the LCOF. They enhanced the Raman signal of benzene and tetrahydrofuran in a hollow quartz fiber. The SEF was 103 times greater than conventional sampling techniques. Total reflection of the light occurred at the sample−glass interface because the refractive index (n) of the organic solvents was larger than that of silica (n = 1.46). In

revious studies have applied vibrational spectroscopy to the analysis of molecular structures, as it takes advantage of the level of sensitivity required to detect structural changes. In the protein sciences, studies have used Raman spectroscopy as a tool to identify changes in the dihedral angle of main1 and side chains2,3 by detecting changes in the intensity and peak position of Raman bands. The ability to record high-quality measurement data is important to ensure the reliability of the derived information. We can assess experimental data quality using the signal-to-noise (S/N) ratio. One of the difficulties associated with Raman spectroscopy is signal detection: Raman cross-section is typically 10−29 cm2 Sr−1 or less,4 and hence signal intensity is generally much weaker than the excitation beam intensity. Despite the difficulty posed by signal detection, Raman spectroscopy is still a valuable and promising method to study (bio)molecules and the chemical analysis of unknown samples because recorded data are directly comparable with a large quantity of accumulated data,5,6 as well as commercial Raman spectra databases. This benefit is not characteristic to other advanced Raman techniques, such as surface-enhanced Raman,7 hyperRaman,8 and nonlinear Raman9 spectroscopy, which yield their own unique spectral patterns. Besides advances in instrumentation,10 the proposals of various sample cell designs have improved the S/N ratio. One method to improve the S/N ratio is to suppress noise via longterm accumulation. Spinning11 or flow cells12−14 are available that reduce sample damage due to photoirradiation. The other © XXXX American Chemical Society

Received: March 22, 2019 Accepted: July 1, 2019 Published: July 1, 2019 A

DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

through holes (a 2 mm hole and a pinhole), and a coverslip.25 The PI film was flanked by the holder made of polyether ether ketone (PEEK) and a coverslip. These parts were tightly glued to each other. The pinhole was formed on the PI film using a laser drilling technique. The sample solution in the reservoir was delivered to the pinhole through the duct in the PEEK holder with a plunger pump (PU-4580, Jasco) and flowed from the pinhole. The flow rate was 4.5 mL min−1. As a stopper of the vertical flow, a glass plate or an Al mirror (25 mm in diameter) was used. A thin strip of a filter paper was put on the stopper to drain the sample solution. By considering a focal point spot size for the excitation laser (fwhm 48 μm; Figure S1), we examined pinholes of 100 μm and larger. The thickness of the PI film was thinner than the pinhole diameter (φ) to avoid vignetting: the 200 and 150 μm pinholes were placed on the 100 μm film and the 125 and 100 μm pinholes were on the 75 and 50 μm films, respectively. Spectrometer. Raman spectra were collected using a spectrometer equipped with a multimode fiber-optic probe (Bayspec). The excitation wavelength was 785 nm (cw), and the laser power at the sample point was 300 mW, unless otherwise specified. The laser power was unchanged before and after passing through the liquid column (data not shown). An aspherical lens (NA 0.64, Thorlabs) was attached to the fiber-optic probe. This lens focused the laser beam onto the pinhole of the VF unit and collected the Raman signal. The collected signal was delivered to the monochromator through the optical fiber (200 μm in diameter). The monochromator was equipped with a transmission grating (970 gr mm−1), and the Raman signal was detected by a charge-coupled device (CCD) detector (1428 × 64 effective pixels; Nunavut, BaySpec) operated at −15 °C. The CCD pixel size was 14 × 14 μm. The spectrometer covered the range from 100 to 2300 cm−1. The width of the entrance slit was 25 μm, which corresponds to a spectral resolution between 8 and 10 cm−1. We performed wavenumber calibration using indene. Peak position reproducibility was ±1 cm−1. For a “conventional” Raman measurement, we tested a 100 μM sample contained within a glass-bottom dish (GBD), as well as a sample placed inside of a quartz cell (4.5 cm in depth). For both samples, signal intensity was identical. Samples. Doubly distilled water was used for the spectroscopic measurement of H2O. BSA was purchased from Sigma-Aldrich and used as received. BSA was dissolved in a 50 mM phosphate buffer (pH = 7.4), supplemented with 100 mM NaCl. Concentrations were set to 0.03, 0.1, 0.3, 1, 3, and 10 mg mL−1 (0.45−150 μM). The fluid volume was set to sufficient value (10 mL) to avoid depletion of the sample liquid. Methanol (Macron Fine Chemicals) was applied to measurements without further purification. Conditions. The Raman spectrum of H2O was measured by averaging three spectra (5 s exposure and 12-fold accumulation for each spectrum). BSA spectra were measured by averaging 20 spectra (4 s exposure and 15-fold accumulation for each spectrum). Methanol spectra were measured by averaging 600 spectra (100 ms exposure for each spectrum). Dead time due to data readout (10 ms) was negligible (∼0.03% of the overall measurement time). Contributions from the coverslip as a glass substrate and the CCD detector dark count were subtracted from the recorded Raman spectra. To compensate for channel-to-channel alterations with respect to CCD detector sensitivity, the

other words, this cell is only suitable for liquids that have a refractive index that is higher than that of silica. Despite its importance in the study of biomolecules, LCOF measurements have not been performed for aqueous solutions. This is because the n for most solid materials is larger than 1.33 (H2O). Partial reflection was applied alternatively; a study demonstrated the 33-fold enhancement of the 1049 cm−1 band for nitrate with a 488 nm Ar+ laser for a 0.1 M KNO3 aqueous solution in a 70 cm capillary glass (1.27 mm i.d.).18 This enhancement was due to partial reflection at the solution−glass interface and total reflection at the glass−air interface. The LCOF for aqueous solutions had not been performed until Altkorn et al. employed a fluoropolymer (AF-2400), which has a refractive index of 1.29.19 They used this material as the clad in the LCOF, which enhanced the Raman signal for aqueous samples. The SEF for the 532 nm-excited Raman signal was as much as 500 with the 1.21 cm fluoropolymer cell.20 Since its development, this fluoropolymer has been widely used for LCOF. The Raman signal for a 0.5−10 nmol L−1 Fe(II)ferrozine complex in an aqueous solution was detected using a 4.47 m LCOF fluoropolymer cell.21 A combination of LCOF with resonance Raman spectroscopy enabled further improvements in sensitivity. For example, a 2.5 × 10−10 M aqueous solution of β-carotene was characterized by enhanced sensitivity.22 At present, only the fluoropolymers of the Teflon AF series23 are available as a clad for the LCOF measurement of aqueous samples among solid materials. A gaseous material, e.g., air (n = 1.00), is available as a clad (air-cladding). The first report of LCOF with the air-cladding was published in 2003 by Spiegel et al.24 They formed the liquid column of the aqueous sample from 50−150 μm pinholes and introduced a 532 nm pulsed beam with Nd:YAG laser coaxially to the liquid column from the end. Highly efficient generation of the stimulated Raman scattering and subsequent decrease in the energy of incident beam were observed, and the total reflection effect at the water−air interface was demonstrated. We have performed the Raman measurement by using the air-cladding LCOF and the microscope objective to couple the incident beam.25 The liquid column of the sample solution was prepared with a flow from a pinhole, and the Raman spectrum was measured at the end point of the liquid column (vertical flow (VF) method). A 12-fold enhancement of the Raman signal has been achieved. Persichetti et al. also reports nearly 1 order improvement of the detection limit (LOD) of ethanol in water by using the aircladding LCOF.26 Furthermore, a recent interesting idea to form a fluidic waveguide is to use a pair of superhydrophobic plates that contained sharp-tip nanostructures.27 It is crucial to optimize the optical and mechanical parameters of the apparatus to maximize the efficiency of the enhancement of Raman signal with the air-cladding LCOF. In this study, we optimized the design of the sampling unit for the VF method to maximize the signal level. We were able to enhance the Raman signal of H2O by up to 90 times. The VF method was applied to the Raman spectroscopic analysis of bovine serum albumin (BSA) in an aqueous solution. The spectrum was measured in solutions with concentrations as low as 0.1 mg mL−1 (1.5 μm), with LOD of 0.029 ± 0.003 mg mL−1 (0.44 ± 0.05 μM).

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EXPERIMENTAL SECTION Sampling Unit. The sampling unit consists of three parts: a holder with a sample duct, a polyimide (PI) film with two B

DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry recorded spectra were divided by a tungsten lamp continuum emission spectrum.

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RESULTS Figure 1(a) shows a diagram of the VF unit. The sample solution in the reservoir infiltrates the gap between the PI film

Figure 2. Raman spectrum of H2O measured by the VF method with a mirror (dotted) and glass plate (solid) and by a conventional method using a glass-bottom dish (broken, 10× magnification). Laminar flow length (l) is described therein. Inset: the dependence of the laminar flow length on SEF with a glass plate (○) and reflection mirror (●) used to block laminar flow. Figure 1. (a) Diagram of the vertical flow unit and (b) characteristic parameters of laminar flow. See text.

and coverslip and flows from the pinhole to form the laminar flow column. The sample column and surrounding air respectively function as the core and clad and allow LCOF sampling.25 The excitation beam was introduced from the pinhole, at which point the Raman signal was collected, delivered to the monochromator, and detected. Considering the refractive indices of air (nclad = 1.00) and H2O (ncore = 1.33), the critical angle for total reflection is 49°. The sample column confines the Raman signals inside the cone that has an apex angle (θapex, Figure 1(b)) of 82° (0.49π Sr in the solid angle). The NA for the LCOF (NALCOF) in air (n = 1.0) and maximum divergence angle (θmax, Figure 1(b)) at the end of LCOF are 0.88 and 122° (1.04π Sr), respectively, and the NALCOF value is calculated with the following equation: NALCOF = n sin θmax =

2 2 ncore − nclad

Figure 3. Dependence of the enhancement factor (SEF) on laminar flow length (l) with different pinhole sizes (φ) ranging from 100 to 200 μm with a glass plate (denoted as φ in the figure) or 100-μm pinhole with mirror (denoted by “0.1 + M”) measured in triplicate. Inset: φ-dependence on d(SEF)/dl. Error bars show the standard deviation of the triplicate measurements.

(1)

The H2O Raman signal was recorded using the VF method with a 100 μm pinhole at different laminar flow lengths (l) (Figure 2, solid traces). The error at each data point was less than 1% of the recorded Raman intensity. We changed l by adjusting the height of an inserted glass plate used to block laminar flow (Figure 1). The Raman signal heightens as l increases. We chose the HOH bending band at 1632 cm−1 and calculated the SEF as a ratio of band intensity, measured by the VF method (IntVF), to the intensity measured by the height (IntGBD): SEF = IntVF/Int GBD

The pinhole size dependence was evaluated with d(SEF)/dl rather than SEF because (i) the absolute value of l was slightly erroneous due to the nonflat nature of the PI film at high pressure, which is a consequence of sample flow, and (ii) the sample−air interface was not completely uniform at both end of the laminar column (at the pinhole and the mirror) because of the turbulence of the sample flow. This error was not a concern by taking into account the differences in l, and therefore the error in l did not affect d(SEF)/dl. The inset in Figure 3 shows the pinhole size dependence on d(SEF)/dl. Consistent with our previous study,25 d(SEF)/dl increased as pinhole size decreased. While d(SEF)/dl is 2.7 ± 0.4 with the 100 μm pinhole and glass stopper (Figure 3, purple), this ratio becomes 5.5 ± 0.1 by replacing the glass plate with the Al mirror (Figure 3, orange). Mirror insertion increased the d(SEF)/dl ratio by 2.0 ± 0.1. The observed mirror effect is possibly explained by considering the reflection of both the Raman signal and excitation beam. On the basis of previous studies, we can expect a 4-fold enhancement of the Raman signal with this configuration,28 because (i) the length of the liquid column (l

(2)

SEF increases from 17.8 to 41.4 as the laminar flow length rises from 6.4 to 13.7 mm (Figure 2, inset, ●). The SEF was also measured by inserting an Al mirror as a stopper (Figure 1). The SEF increases proportionally with l (Figure 2, dotted traces), i.e., SEF increases from 51.3 to 90.2 as l rises from 6.2 to 13.1 mm. The slope in a plot of SEF against l [d(SEF)/dl] (Figure 2, inset) shows that the SEF increases by 3.1 as laminar flow lengthens 1 mm. The slope is 5.6 (open circle) and increases by nearly 2-fold with the mirror. Figure 3 shows the SEF−l plots with different pinhole sizes. The measurement was performed thrice for each condition. C

DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry in eq 3) is doubled, and (ii) the mirror reflects the Raman signals that direct away from the pinhole. The reflection of the leaving Raman signal makes the signal intensity twice larger. The four-fold enhancement was anticipated for this reason. The observed 2-fold improvement suggests that the reflection of each component was not complete. The confined light dissipated from the laminar column at the stopper because it most likely deformed the cylindrical shape of the laminar column. The intensity of the Raman signal increases as the power of the excitation beam increases from 25 to 300 mW (Figure 4).

Figure 4. Laser power dependence for the H2O Raman intensity spectrum. Laser power is 300 (top), 250, 200, 150, 100, 50, and 25 (bottom) mW, respectively. Pinhole size is 100 μm. Inset shows the laser power dependence for the peak intensity at 1632 cm−1.

The peak height at 1632 cm−1 linearly depends on laser power (Figure 4, inset). The observed linear dependence indicates the occurrence of linear optical processes, i.e., spontaneous Raman scattering, rather than nonlinear processes, such as stimulated Raman scattering that occurs in micron-sized liquid droplets.29 Besides this, SEF (=80) was constant for the entire range in the Raman spectrum of methanol (Figure 5). We did not detect a wavenumber dependence of SEF.30 BSA is an α-helix-rich globular protein (cf. PDB ID 3V03). The structures of the main and side chains are illustrated in Figure 6, parts a and b, respectively. This protein consists of 583 residues connected with the N−Cα−C(O) linkage of the peptide main chain (Figure 6(a)) and involves 2 Trp, 20

Figure 6. Main chain (a) and side chain (b) structures of BSA. The Raman spectra of BSA measured using the VF method at 10 (red), 3 (yellow), 1 (green), 0.3 (blue), 0.1 (purple), and 0.03 (black) mg mL−1. Intensity is linear (c) and normalized (d). In part a, the numbers denote the peak position of the BSA signals, and * indicates a buffer band.

Tyr, 27 Phe, and 17 S−S bridges (shown by green, magenta, gray, and cyan, respectively, in Figure 6(b)). Figure 6(c) shows the Raman spectrum of BSA measured with the VF method. The solvent band was subtracted such that the 1700−1800 cm−1 region became flat. We observed several BSA Raman bands31,32 at 1650 (amide I), 1445 (the scissoring of CH2 and CH3 groups), 1330 (the CH2 wagging and the Trp doublet), 1002 (the ring breathing mode of Phe), 937 (the N−Cα−C stretching of the main chain in the α-helix), 849 and 825 (the Tyr doublet), and 507 cm−1 (the S−S stretching). Figure 6(d) shows the spectra for which intensity is normalized by concentration for comparison. The three BSA peaks are commonly observed between 0.3 and 10 mg mL−1 (4.5−150 μM). The estimated S/N ratio is 300, 15, and 5 for results obtained at concentrations of 10, 0.3, and 0.1 mg mL−1, respectively, and worsens below 0.1 mg mL−1 (1.5 μM). The three peaks were not observed at 0.03 mg mL−1 (0.45 μM). The peak intensity sum for the three bands at 1650, 1445, and 1330 cm−1 are plotted against sample concentration (Figure 7; see Supporting Information for data analysis details). Figure 7 also shows the fit of the regression line to the intensity plot against concentration. Signal intensity increases proportionally with sample concentration. The regression line reproduces the

Figure 5. Raman spectrum of methanol measured by the VF (solid line) and conventional methods (dotted line; 80-fold enhanced). Pinhole diameter was 100 μm, laminar flow length was 15 mm, with the reflection mirror, and the flow rate was 2 mL min−1. D

DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

ÅÄÅ ÑÉÑ n ÅÅij dσ yz ∼ −4 Ñ ∼ Å j z s = ÅÅj (ν − νk) ÑÑÑÑ(∼ ν −∼ νk)4 N R ÅÅÇk dΩ z{k ref ÑÑÖ L n0 (n 2 + 2)2 (n02 + 2)2 × R 34

Analytical Chemistry

observed linear dependence (R2 = 0.999) in each of the measurements performed thrice. The limit of detection (LOD) and qualification (LOQ), calculated from the 3- and 10-fold values for intercept standard deviation (as the SD of the blank value) divided by the slope, were 0.029 ± 0.003 and 0.095 ± 0.008 mg mL−1 (0.44 ± 0.05 μM and 1.43 ± 0.12 μM), respectively. The LOQ is consistent with the fact that the three BSA peaks appear at 0.1 mg mL−1 (Figure 6b).

spot size = c ×

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2 nCO

0.22 NA obj

(5)

where c is the fwhm of the excitation beam diameter at the end of the optical fiber. Here we calculated c as 167 μm based on the experimental value of fwhm (48 μm) at an NAobj value of 0.64 (Figure S1). There was 100% light energy penetration through the pinhole at the aperture and 0% at the other points in this simulation. The throughput increases with larger NAobj (as the focal spot size decreases) and φ values and vice versa (Figure 8(a)). For the second point, the sample column confines light when the incident angle to the sample−air interface is larger than the critical angle (49°). This leads to an incident beam that partly dissipates when NAobj exceeds 0.88 (Figure 8(b)).

DISCUSSION The Raman signal of H2O was enhanced by up to 90-fold using the VF method (Figure 2). The 100 μm pinhole had superior performance in our experiments, and the mirror at the end of laminar flow enhanced signal intensity 2-fold (Figure 3). The SEF increased as laminar flow became longer (Figure 4) and was independent of wavenumber (Figure 5). We successfully measured the BSA spectrum even at 0.1 mg mL−1 (1.5 μM) (Figure 6). The LOD and LOQ were 0.029 ± 0.003 (0.44 ± 0.05 μM) and 0.096 ± 0.008 mg mL−1 (1.44 ± 0.12 μM), respectively (Figure 7). Hence, we have developed and verified a simple technique to improve Raman spectrum signal intensity using universal materials. The VF method allows us to record nonresonance Raman signals with concentrations that are 101−102-fold lower than the conventional method (in a range of 100 μM33−35). The signal enhancement effect in our study should be attributed to the improved efficiency of the signal collection because effects due to changes in the molecular parameters including the Raman scattering crosssection cancels in the comparison of the Raman signal intensity using the vertical flow and the conventional methods. A “short LCOF” model by Altkorn et al. explains the observed linear dependence that SEF has on l (Figure 3): LCOF is short enough that light attenuation is negligible in this model. The Raman power, PR, at the face of LCOF in air is given by the following equation:36 PR =

(4)

where (dσ/dΩ)k is the differential Raman cross-section (Sr−1 cm2) of the kth vibration, ν̃ref is an excitation frequency (cm−1) used to measure (dσ/dΩ)k, ν̃k is the vibrational frequency (cm−1) of the kth vibration, ν̃L is the frequency of the excitation laser, n0 and nR are the sample refractive indices at the exciting and scattered radiation frequencies (n0 and nR are nearly equal to nCO in our experiment),36 and N is the number density of analyte molecules (cm−3). Equation 3 does not account for the φ dependence on IntVF (or SEF or d[SEF]/dl) that we observed in Figures 2 and 3. We simulated the dependence of signal intensity on φ and NA of the objective lens (NAobj) by considering excitation beam and Raman signal separately. The dependence of excitation beam throughput on φ and NAobj appears at the pinhole and total reflection condition. For the first case, focal point spot size is the ratio of NAobj to NA of the optical fiber that delivers the excitation beam (0.22), calculated with the following equation:

Figure 7. Plots of peak height against the sample concentration in three measurements. Dashed line shows a regression line.

2 2 PLsπ (nCO − nCL )

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Figure 8. Signal intensity dependence on NA and pinhole size (φ) due to (a) incident energy throughput at the pinhole, (b) energy loss due to total reflection, (c) collection lens surface coverage, and (d) coupling efficiency at the optical fiber. Overall effect is plotted in part e. The maximum value of signal intensity is normalized to unity in parts a−e. (f) A comparison of the observed φ dependence on d[SEF]/dl (left axis) and calculated signal intensity throughput at NA of 0.64 (right axis; overall, solid; at the pinhole, dashed; coupling efficiency, dotted).

l (3)

where PL is the laser power, nCO and nCL are the refractive indices of the core and clad of the LCOF, respectively, l is the length of the LCOF, and s is the Raman scattering coefficient (Sr−1 cm−1) defined as37 E

DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

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28° (0.06π Sr) in the LCOF using the fluoropolymer (nclad = 1.2930). On the basis of the θapex values, the VF method has the potential to collect Raman signals eight times more efficiently per unit length.

On the other hand, the dependence of the Raman signal throughput on φ and NAobj is seen in the signal collection efficiency at the end of the LCOF and in the coupling between the LCOF and optical fiber. For the first case, the Raman signal is not fully collected when NAobj is smaller than 0.88 because the objective lens does not cover the divergence angle (θmax) at the end of LCOF. The coverage (defined as a value between 0 and 1) was calculated based on the following equation: 2π (1 − coverage =

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CONCLUSION In this study, we have refined the VF method by optimizing the design of the sampling unit and succeeded to enhance the intensity of the Raman signal of H2O by up to 90 times. The pinhole size dependence of the enhancement factor has been ascribed to that of the coupling efficiency of the collected signal at the LCOF and optical fiber. In the Raman analysis of BSA in the aqueous solution, the spectrum has been measured with the concentrations as low as 0.1 mg mL−1 (1.5 μm), with LOD of 0.029 ± 0.003 mg mL−1 (0.44 ± 0.05 μM); it has become possible to record the nonresonance Raman signals with concentrations 101−102-fold lower than previously. The VF method is applicable not only to nonresonance Raman spectroscopy but also to other optical experiments, such as advanced Raman and emission spectroscopies, e.g., fluorescence and Rayleigh scattering. The VF method provides an efficient sampling technique to enhance signal intensity with universal materials.

2 1 − NA obj )

2π (1 − cos θmax )

(6)

to obtain the NAobj dependence (Figure 8(c)). With respect to the second point, the pinhole diameter from an image at the terminal of an optical fiber (φ′) was (NAobj/0.22)-times as large as φ when the NA of the imaging lens was 0.22: ϕ ′= ϕ ×

NA obj 0.22

(7)

The Raman signal from the LCOF is delivered entirely to the coupled optical fiber (200 μm in diameter) when φ′ is equal to or smaller than 200 μm. This is not the case when φ′ is larger than 200 μm.36 The efficiency (defined as a value between 0 and 1) is given by the following equation: π (200 μm)2 efficiency = πϕ′2

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.9b01472. Determination of focal spot size and details of the data analysis (PDF)

(8)

Thus, the coupling efficiency is a function of NAobj and φ (Figure 8(d)). The overall dependence of IntVF on φ and NAobj is eventually obtained as shown in Figure 8(e). The φ dependence of IntVF is equivalent to that of SEF (and d[SEF]/ dl) because IntGBD is independent of φ. The observed φ dependence of d[SEF]/dl (Figure 3) is consistent with the calculated plot at NAobj = 0.64 (Figure 8(f), solid line). The excitation beam throughput at the pinhole (Figure 8(a); broken line in Figure 8(f)) and the coupling efficiency at the LCOF and optical fiber (Figure 8(d); dotted line in Figure 8(f)) caused the φ dependence of the signal intensity. The latter factor explains the observed φ dependence of d[SEF]/dl. The wavelength-dependent decrease in SEF was observed during the 785 nm-excited Raman spectrum measurement of MeOH using an LCOF with the AF-2400 fluoropolymer: SEF was 37 at 1033 cm−1, 22 at 1450 cm−1, and 9 at 2834 and 2943 cm−1, with the 393 cm long and 150 μm i.d. LCOF.30 The dependence was explained as a consequence of the selfabsorption due to the overtone bands in the near-IR region.30 This explanation is plausible because the alcohol absorption bands at 12 000 cm−1 (assignable to the third-overtone of the C−H stretching band)38,39 attenuate the Raman signal intensity of the CH3 bending band at 1450 cm−1 (11 300 cm−1). The attenuation and resultant wavenumber dependence of SEF was not observed in our experiment (Figure 5). However, this is most likely due to the fact that absorbance in the near-IR region was not large (the absorbance is ∼0.2 when measured with a 6 cm cell)39 and the length of the LCOF was short (15 mm). We also note that the SEF using the VF method (40 with a 100 μm pinhole without the mirror) (Figure 3) was comparable to that of the LCOF with AF-2400 (SEF = 9−37)30 although the lengths of the LCOF were quite different from each other (14 mm and 393 cm, respectively). The critical angle was 49° and θapex was 82° (0.49π Sr) in the VF method, whereas the critical angle was 76° and θapex was

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +886-3-5712121 (ext 56540). ORCID

Hirotsugu Hiramatsu: 0000-0002-5239-3032 Author Contributions

The manuscript was written through contributions of all authors. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by Ministry of Science and Technology, Taiwan (MOST 107-2113-M-009-012-MY2), and by the Center for Emergent Functional Matter Science of National Chiao Tung University from the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.

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REFERENCES

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DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.9b01472 Anal. Chem. XXXX, XXX, XXX−XXX