Laser desorption/ionization mass spectrometry of L-thyroxine (T4) using combi-matrix of α-cyano-4-hydroxycinnamic acid (CHCA) and graphene

An optimal combi-matrix for MALDI-TOF mass spectrometry was presented for the analysis of L-thyroxine (T4) in human serum. For the selection of the optimal combi-matrix, several kinds of combi-matrices were prepared by mixing the conventional organic matrix of CHCA with nanomaterials, such as graphene, carbon nanotubes, nanoparticles of Pt and TiO2. In order to select the optimal combi-matrix, the absorption at the wavelength of laser radiation (337 nm) for the ionization of sample was estimated using UV–Vis spectrometry. And, the heat absorption properties of these combi-matrices were also analyzed using differential scanning calorimetry (DSC), such as onset temperature and fusion enthalpy. In the case of the combi-matrix of CHCA and graphene, the onset temperature and fusion enthalpy were observed to be lower than those of CHCA, which represented the enhanced transfer of heat to the analyte in comparison with CHCA. From the analysis of optical and thermal properties, the combi-matrix of CHCA and graphene was selected to be an optimal combination for the transfer of laser energy during MALDI-TOF mass spectrometry. The feasibility of the combi-matrix composed of CHCA and graphene was demonstrated for the analysis of T4 molecules using MALDI-TOF mass spectrometry. The combi-matrix of CHCA and graphene was estimated to have an improved limit of detection and a wider detection range in comparison with other kinds of combi-matrices. Finally, the MALDI-TOF MS results of T4 analysis using combi-matrix were statistically compared with those of the conventional immunoassay.


Introduction
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used to analyze biomolecules such as proteins and deoxyribonucleic acid (DNA) with a high molecular weight (Karas and Hillenkamp 1988;Tanaka et al. 1988;Guinan et al. 2015;Lu and Cai 2012). Organic matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) are used to absorb the laser radiation in the ultra-violet (UV) range and as proton sources to ionize analytes (Leopold et al. 2018;Bateson et al. 2011). The organic matrices are fragmented during laser energy absorption. These fragments produce mass peaks in the low m/z ratio range of less than 1000 (Kim et al. 2014a;Kim et al. 2013;Park et al. 2015;Park et al. 2017). Because the mass peaks from organic matrices are unreproducible, it is difficult to identify the mass peaks of analytes that occur at low m/z ratios (Noh et al. 2021;Kim, et al. 2021a;Kim, et al. 2021). Therefore, the capability to analyze small molecules is minimal. Organic matrix and analyte formed a crystal with heterogeneous morphology after drying process on a metal plate for MALDI-TOF MS analysis. Depending on the position of the laser radiation, different amounts of analyte ions occur even for the crystals of the same concentration. Thus, the quantitative analysis of small molecules has been restricted by MALDI-TOF MS (Park et al. 2019;Kim et al. 2017;Kim et al. 2020;Choi et al. 2018;Noh et al. 2017).
Matrix selection in MALDI is critical in determining the success or failure of the analysis (Zenobi and Knochenmuss 1998). Various solid matrices composed of inorganic materials have been reported as alternative matrices to solve the problems of the quantitative analysis of small molecules and extend the application range in addition to enhancing the sensitivity of MALDI-TOF MS. These solid matrices are fabricated to produce nanostructures such as nanoparticles (NPs), nanotubes, and nanowires. The solid matrices can be classified according to the base materials: (1) carbon-based nanostructures such as graphene (Banazadeh et al. 2018;Gulbakan et al. 2010;Lu et al. 2011;Chen et al. 2013;Liu et al. 2015;Oh and Biswas 2021), carbon nanotubes (CNTs), and fullerene (Szabo et al. 2010); (2) semiconductor-based nanostructures such as TiO 2 (Kim et al. 2016;Kim et al. 2018;Kim et al. 2019;Kim et al. 2014b;Chen and Chen 2004;Kim and Lee 2021), SnO 2 (Shen, et al. 2010), and CdS (Shrivas and Wu 2010;An et al. 2021); (3) metal-based nanostructures such as Au (Chen et al. 2010;Nayak and Knapp 2010;Kawasaki et al. 2012;Nguyen et al. 2016;Ly and Joo 2014;Li et al. 2012;Kim, et al. 2021b), Pt (Navin et al. 2009), and Ag (Guan et al. 2018). Usually, carbon-based nanostructures can be used as solid matrices because they can absorb laser energy in the UV range and be used as a proton source (Park et al. 2018;Lu et al. 2017). Semiconductor-based nanostructures can also absorb the laser radiation energy and create holes in the valence band and electrons in the conduction band, which can be used to ionize analytes through oxidation and reduction. Metal-based nanostructures can also absorb the laser radiation energy and effectively increase the surface temperature from low thermal conductivity to enhance the ionization of the analyte. Among them, graphene has compelling properties such as strong optical absorption at laser wavelengths, high thermal conductivity (approximately 5000 W mK −1 ), and fast electron mobility with efficient electron-phonon coupling, making graphene one of the most promising materials for mass spectrometric applications (Balandin et al. 2008;Bolotin et al. 2008;Chung et al. 2013;Kim et al. 2011).
L-thyroxine (T 4 ) is a thyroid hormone secreted from the thymus that regulates growth and development, carbohydrate metabolism, and protein synthesis (Sinha et al. 2014;Mullur et al. 2014). Monitoring T 4 in human serum and tissues is necessary to assess overall health. For example, a medical diagnosis of congenital hypothyroidism was performed using a cutoff T 4 concentration of approximately 50 ng mL −1 (LaFRANCHI 1999;Desai et al. 1994). Clinical laboratories measure the T 4 concentration by radioimmunoassay (RIA), which requires the handling and disposal of radioactive materials (Mizuta et al. 1982;Oberkotter and Tenore 1983;Bong et al. 2021;. In this study, a new combimatrix will be presented for the effective ionization of the T 4 , which has a low ionization efficiency in MALDI mass analysis. A combi-matrix composed of a conventional CHCA and solid matrix will be presented for the quantitative analysis of T 4 . The concept of a combi-matrix is to: (1) enhance the absorption of UV radiation energy and (2) transfer it for the thermal desorption of analytes by mixing solid materials, as shown in Fig. 1a. Ultraviolet-absorbing materials such as CNTs, Pt NPs, and P25 (TiO 2 ) with high thermal conductivity (Table 1) will be tested as solid matrices to optimize the combi-matrix for the quantitative analysis of T 4 , which can effectively absorb the laser radiation to enhance the desorption process (Choi et al. 2018;Banazadeh et al. 2018;Rodríguez et al. 2015;Chen et al. 2016). As shown in Fig. 1b, the mixing of CHCA and a solid matrix changes the ionization efficiency of T 4 . The absorption properties of the combi-matrices will be analyzed by wavelength scanning around the UV radiation (337 nm), and the thermal properties to transfer the absorbed energy will be analyzed by differential scanning calorimetry (DSC) to optimize the combi-matrix for T 4 analysis. Quantitative analysis of T 4 will be conducted for T 4 samples in human serum using each combi-matrix.
Combi-matrix solutions of CHCA and graphene, CNT, P25, and Pt NPs were prepared by mixing the solutions at volume ratios of 1:1. The T 4 sample was premixed with a combi-matrix of CHCA and graphene (1:1 v/v). The mixture (1 μL) was spotted on a stainless-steel target plate and dried for MALDI-TOF MS analysis.
Human serum was spiked with T 4 standard samples to demonstrate the feasibility of diagnosing hypothyroidism. Protein precipitation or extraction was conducted before mass analysis using various organic solvents to precipitate numerous proteins in serum samples (Alshammari et al. 2015). Methanol precipitation was conducted by mixing serum samples (50 μL) with cold methanol (450 μL) and incubating for 20 min. Samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was used for the MALDI-TOF MS analysis. The yield (extraction efficiency) of each protein precipitation method was compared by calculating the recovery at 50 ng mL −1 (the cutoff concentration for hypothyroidism).

Characterization of the combi-matrix
The size and distribution of the combi-matrix of CHCA and graphene, CNT, Pt NPs, and P25 were characterized  Table 1 Thermal properties of the solid matrices

P25 Pt NPs CNTs Graphene
Thermal conductivity (W/ (m•K)) 0.61 100-500 2,400 4,500-5,000 Electron mobility (cm 2 /(V•s)) 10 -4 -10 -5 10 0 80,000 200,000 Effective surface area (m 2 /g) 50 72 380 700 Page 4 of 15 Noh et al. Journal of Analytical Science and Technology (2022) 13:5 by using transmission electron microscope (JEM-F200, JEOL, Japan). The optical properties of the combi-matrices were analyzed by using UV/VIS spectrometer (Perkin Elmer, Massachusetts, USA). The thermal properties of the combi-matrices were analyzed through differential scanning calorimeter (PerkinElmer, Massachusetts, USA). The heat flow for CHCA and the combi-matrix was observed in the temperature range of 200-300 °C at an increasing rate of 15 °C/min. Asparagine (Asn, melting temperature of 235 °C) was used as a model heat sink that absorbed the heat flow from the combi-matrix to melt. The thermal properties, onset temperature (T onset ), peak temperature (T peak ), fusion enthalpy (ΔH fus ), and relative thermal conductivity were estimated from the DSC spectrum (Kim et al. 2019).

MALDI-TOF MS analysis
Mass spectrometric analysis of the T 4 standard was conducted using a Voyager DE-STR (AB Sciex, Foster City, CA, USA) equipped with a 337-nm wavelength N 2 laser. The mass spectra were obtained using the positive ion mode with an accelerating voltage of 20 kV and a second ion source voltage of 18 kV. The reflector voltage was set to 9.5 kV, and all mass spectra were obtained by integrating 300 laser pulses for each sample. The signal-tonoise ratio (S/N) was adopted from the equation in the reference (Müller et al. 2001). Ion peaks with S/N larger than three were considered mass signals. The measured intensities for each concentration were plotted as graphs and linearly approximated using the least-squares method. The calculated limit of detection was three times the noise level. The reproducibility of MALDI-TOF MS was verified by inter-and intra-spot analyses of T 4 at a concentration of 200 ng mL −1 . Inter-spot analysis was performed at five different sample spots of T 4 , and the measurement was taken using the partial random walk function. Intra-spot analysis was also conducted at the same sample spot of T 4 through five rounds of repeated measurements using the partial random walk function.

Statistical analysis
MedCalc software for Windows version 18.6 (Ostend, West Flanders, Belgium) was used for all statistical analyses. The MALDI-TOF MS data were expressed as the mean ± standard deviation. The Mann-Whitney unpaired test was used to examine differences between the MALDI-TOF MS data of only CHCA and the combimatrices of CHCA and graphene, CNTs, Pt NPs, and P25. A conventional immunoassay was performed as a reference method to analyze free T 4 in the serum at different concentrations using a commercially available ELISA kit for free T 4 from Wkea (Changchun, Jilin, China). The Bland-Altman plot and Passing-Bablok regression were employed to confirm the coincidence of the two analysis methods of MALDI-TOF MS based on the combi-matrix and immunoassay.

Properties of combi-matrix
The concept of a combi-matrix was (1) to enhance the absorption of UV radiation energy and (2) to transfer it for the thermal desorption of analytes by mixing solid materials, as shown in Fig. 1a. The organic matrix of CHCA is mixed with carbon-based nanostructures of graphene, CNTs, Pt, and P25 with high thermal conductivity to prepare the combi-matrix, as summarized in Table 1. First, the solid matrices of graphene, CNT, Pt NPs, and P25 were utilized to ionize T 4 molecules without any organic matrix. For the comparison of mass peaks, the concentration of solid matrices was set at 0.5 mg mL −1 , and the same amount of solid matrix was used by dropping the same volume (1 µL, the absolute amount of 500 ng) for analyzing T 4 . As shown in Fig. 1b, the intensity of the mass peak (S/N ratio) of the T 4 molecule (at a concentration of 200 ng mL −1 ) was 237.5 (2.7), no signal (− 0.5), 5.2 (− 0.4), and 19.9 (− 0.2) for graphene, CNTs, Pt NPs, and P25, respectively. Under similar conditions, the mass peak from the analysis using CHCA was 1248.5 (16.5). The results suggested that the intensity of the mass peak was significantly lower than that of CHCA when only solid matrices were used for ionizing T 4 molecules. After mixing CHCA with the solid matrices, the intensity of the mass peaks (S/N ratio) of T 4 molecules was 3341.8 (45.0), 2093.8 (28.0), 1650.6 (23.6), and 1513.8 (24.5) for graphene, CNTs, Pt NPs, and P25, respectively. The results indicated that the mass intensity and the S/N ratio were significantly increased after mixing CHCA with solid matrices of graphene, CNTs, Pt NPs, and P25. Furthermore, the mass peaks of the combimatrices consisting of CHCA and solid matrices of graphene, CNTs, P25, and Pt NPs were higher than those of the conventional organic matrix of CHCA. Additionally, the mass peak intensity of different combi-matrices coincided with the order of thermal conductivity and the effective surface area of used solid matrices, as summarized in Table 1 (Grinou et al. 2012;Choo et al. 2012;Kim and Park 2007;Sikdar et al. 2011). Combi-matrix of graphene and graphene showed the highest mass intensity, as graphene showed the highest thermal conductivity (4500-5000 W/(m K)), electron mobility (200,000 cm 2 / (V s)), and effective surface area (700 m 2 /g) among the solid matrices. Thus, the concept of a combi-matrix: (1) to enhance the absorption of UV radiation energy and (2) to transfer it for the thermal desorption of analytes by mixing solid materials was feasible. The optical properties before and after mixing the solid matrices with CHCA were analyzed by UV/Vis spectrometry to estimate the enhancement of the absorption of UV radiation energy. As shown in Fig. 2a, CHCA and graphene have maximum absorptions at wavelengths (λ max ) of 350 and 270 nm, respectively, which was also reported in previous studies (Robinson et al. 2017;Low et al. 2004;Uran et al. 2017). When graphene was mixed with CHCA, the maximum absorption was observed at a wavelength (λ max ) of approximately 285 nm; the absorption increased at a wavelength of 337 nm of the laser radiation for the ionization of the sample. As the turbidity of graphene and the mixture of CHCA and graphene were controlled to be the same at a wavelength of 500 nm, the amount of graphene was similar before and after mixing with CHCA. However, the absorption at wavelengths less than 350 nm was significantly higher than that of graphene before mixing with CHCA. These results indicated that the absorption of the combi-matrix achieved a significantly increased absorption of laser radiation for the effective ionization of T 4 molecules. The combi-matrix of DHB and graphene demonstrated an identical trend of increased absorbance of laser radiation at a wavelength of 337 nm, as shown in Fig. 2b. The UV/ Vis spectra of 9-AA showed two broad peaks in the wavelength range of 200 to 500 nm. The absorbance at 337 nm increased when graphene was mixed with 9-AA matrix, as shown in Fig. 2c. Compared with the UV/VIS spectra, the enhancement of absorption of laser radiation at the wavelength of 337 nm was estimated to increase by 9.7%, 7.1%, 6.1%, and 3.1% for graphene, CNTs, Pt NPs, and P25, respectively (Additional file 1: Fig. S1). The results suggested that the combi-matrix enhanced the absorption of UV radiation energy during MALDI-TOF MS, and graphene was the optimal solid matrix for the combimatrix formation.
Next, the enhancement of the thermal properties of the combi-matrix for the transfer of laser radiation energy was estimated using DSC. The T onset , T peak , and ΔH fus are estimated from the DSC spectrum through the measurement of heat flow in the controlled temperature range to analyze the heat absorption property, as shown in Fig. 3a. The heat flow was observed for CHCA and the combi-matrix of CHCA and graphene in the temperature range of 200-300 °C at an increasing rate of 15 °C/min, and Asn was used as a model heat sink to absorb the heat flow from the combi-matrix to melt. From the slope of the DSC heat flow curve, the relative thermal conductivity was estimated, representing the amount of heat flow required at the phase change of the heat sink.
The DSC spectra were measured for Asn only, Asn with graphene, Asn with CHCA, and Asn with combi-matrix composed of CHCA and graphene. As shown in Fig. 3b, the (ΔH fus ) of Asn significantly decreases compared to Asn only, Asn with graphene, and Asn with CHCA. The results implied that Asn melted with a lower heat flow by mixing the organic matrix (CHCA) with a solid graphene matrix. Additionally, from comparing the slope of the DSC spectra, the combi-matrix had a lower relative thermal conductivity than other conditions, indicating a more effective transfer of thermal energy to the heat sink material. The same DSC measurements are taken using DHB and 9-AA as organic matrices of the combi-matrix, as shown in Fig. 3c and Fig. 3d. For the two organic matrices, the ΔH fus of Asn and the relative thermal conductivity from the slope of the DSC spectrum were significantly reduced by mixing graphene compared to those of Asn only, Asn with graphene, and Asn with organic matrix. Thus, the combi-matrix composed of the conventional organic matrix and graphene transferred the energy from UV radiation, which effectively transferred to the analyte for MALDI-TOF MS.
When three different kinds of organic matrices are mixed with graphene, the ΔH fus of Asn and the relative thermal conductivity are minimum for the organic matrix of CHCA, as shown in Fig. 3e and f. These results indicated that the optimal combi-matrix was composed by mixing the conventional organic matrix of CHCA and graphene for the energy transfer from UV radiation for MALDI-TOF MS.
The combination of the solid matrix of graphene and the organic matrices of CHCA, DHB, and 9-AA was also applied for the MALDI-TOF MS of Asn. For the MALDI-TOF MS, the Asn sample at a concentration of 100 μg mL −1 was dropped to the combi-matrices of organic matrix and graphene. The mass peak of Asn was observed at the m/z ratio of [Asn + H] + = 133.1 [reflective positive (RP) mode] and [Asn-H] − = 131.1 [reflective negative (RN) mode]. When graphene is mixed with the organic matrix of CHCA, the mass peak of Asn has a significantly high S/N ratio of 4.9, comparison to Asn only (S/N ratio of 0.5), Asn with graphene (0.7), and Asn with CHCA (2.7), as shown in Fig. 4a. Furthermore, when DHB and 9-AA were used as the organic matrix of the combi-matrix with graphene, the S/N ratio was significantly higher than Asn only, Asn with graphene, and Asn with organic matrix. The S/N ratio of the mass peak of Asn was 4.9 for CHCA (Fig. 4a), 3.3 for DHB (Fig. 4b), and 3.3 for 9-AA (in RN mode, Fig. 4c) when the mass peak of Asn from the combi-matrices of different organic matrices. From the previous results, the optimal combimatrix was composed by mixing the conventional organic matrix of CHCA and graphene to absorb the laser energy from the UV/Vis spectrum and the transfer of energy from DSC analysis. These results confirmed that the combi-matrix with graphene and CHCA enhanced the

Analysis of L-thyroxine (T 4 ) using the combi-matrix
The combination of the solid matrix of graphene and the organic matrices of CHCA, DHB, and 9-AA was also applied for MALDI-TOF MS of T 4 . For the MALDI-TOF MS, the T 4 sample at a concentration of 200 ng mL −1 was dropped on the combi-matrices of the organic matrix and graphene. The mass peak of T 4 was observed at the m/z ratio of [T 4 + H] + = 777.8 (RP mode) and [T 4 -H] − = 775.8 (RN mode). When graphene was mixed with the organic matrix of CHCA, the mass peak of T 4 had a significantly higher S/N ratio of 20.6 than T 4 only (S/N ratio of − 0.1), T 4 with graphene (0.8), and T 4 with CHCA (7.8). For the mass peak of T 4 from the combimatrices of different organic matrices, the S/N ratio of the Asn mass peak was 20.6 CHCA [ Fig. 5a], 18.9 for DHB [ Fig. 5b], and 1.6 for 9-AA [in RP mode, Fig. 5c]. These results confirmed that the combi-matrix with graphene and CHCA was optimal for MALDI-TOF MS. Quantitative analysis of T 4 molecules was performed using a combi-matrix organic matrix (CHCA) and a solid matrix (graphene). The inhomogeneous co-crystallization of the matrix analyte made quantitative analysis difficult, and the feasibility of the quantitative analysis of T 4 was demonstrated by investigating the reproducibility of the inter-and intra-spot measurements. Five different sample spots are independently detected, as shown in Additional file 1: Fig. S2a and Fig. 6a to measure the inter-spot reproducibility. Based on the analysis of the mass spectra, the inter-spot reproducibility was 6.7% for T 4 (200 ng mL −1 ). MALDI-TOF mass spectra were obtained at five different positions in one sample spot to estimate the intra-spot reproducibility, as shown in Additional file 1: Fig. S2b and Fig. 6b. Based on the mass spectra, the intra-spot reproducibility was 4.6% for T 4 (200 ng mL −1 ). Therefore, the analysis of T 4 molecules was conducted with high inter-and intra-spot reproducibility using MALDI-TOF MS based on the combi-matrix of organic matrix and solid matrix.
A combi-matrix consisting of CHCA and graphene was used for quantitative analysis of the T 4 molecules. T 4 samples at the concentration range of 12.5-200 ng mL −1 were prepared by considering the cutoff concentration of 50 ng mL −1 for the diagnosis of congenital hypothyroidism (Desai et al. 1994), as shown in Fig. 6c. The standard curve for analyzing T 4 molecules was prepared from the repeated measurement and the mass peak, demonstrating high linearity (r 2 = 0.971) at the corresponding concentration range. Therefore, the quantitative analysis of T 4 was feasible with high linearity in the concentration range, including the cutoff concentration of 50 ng mL −1 , for diagnosing congenital hypothyroidism.
The combi-matrix consisting of CHCA and solid matrices of graphene, CNTs, Pt NPs, and P25 was compared for the quantitative analysis of T 4 molecules at the concentration range of 12.5-200 ng mL −1 (Additional file 1: Fig. S3). Figure 6d shows the test results with four different combi-matrices from the quantitative analysis of T 4 molecules. The combi-matrix of CHCA and graphene had the widest intensity range than the conventional organic matrix of CHCA and other combi-matrices. From the statistical analysis (Mann-Whitney unpaired test), the test results between the organic matrix of CHCA and the combi-matrices of CHCA and graphene, CNTs, Pt NPs, and P25 were independent, with P-values less than 0.0001. Whiskers show the minimum and maximum values, the boxes represent 25-75% data ranges, horizontal lines within boxes are medians, and diamond symbols are the means of the mass spectrometric results. These results showed that the combi-matrix of CHCA and graphene was effectively used for the quantitative analysis of T 4 molecules in a wider concentration range compared to CHCA and other kinds of combi-matrices. T4 molecules were detectable in the low concentration range (below 75% range) when using CHCA and graphene as a combi-matrix from the box plot.
As shown in Fig. 7a, T 4 samples at a concentration range of 12.5-200 ng mL −1 are prepared in human serum after methanol extraction to precipitate proteins, and the extracts of T 4 samples were analyzed using the combimatrices. When T 4 analysis was performed using only CHCA as a matrix, the mass peak of T 4 was observed at a concentration of more than 100 ng mL −1 . When the combi-matrix consisting of CHCA and graphene was used for analyzing T 4 , the mass peak of T 4 was observed over the entire concentration range, including the cutoff concentration of 50 ng mL −1 for diagnosing congenital hypothyroidism. As shown in Fig. 7(b), the standard curve for analyzing T 4 molecules is obtained from the repeated measurements and the mass peaks, exhibiting high linearity (r 2 < 0.99) at the corresponding concentration range. These results indicated that the combi-matrix consisting of CHCA and graphene was used for the quantitative analysis of T 4 samples spiked in serum.
(See figure on next page.) Fig. 3 Analysis of the thermal properties of the combi-matrix. a Schematic view of differential scanning calorimetry (DSC). DSC profiles of the combi-matrix composed of b α-cyano-4-hydroxycinnamic acid (CHCA) and graphene, c 2,5-dihydroxybenzoic acid (DHB), and graphene, and d 9-AA and graphene. The thermal parameters of the onset temperature, peak temperature, enthalpy of fusion, and relative thermal conductivity were estimated from each DSC profile Noh et al. Journal of Analytical Science and Technology (2022)  Page 9 of 15 Noh et al. Journal of Analytical Science and Technology (2022) 13:5 The box plot shows the test results with four different combi-matrices from the quantitative analysis of T 4 molecules, indicating that the combi-matrix of CHCA and graphene had the widest intensity range than the conventional organic matrix of CHCA and other kinds of combi-matrices [ Fig. 7c]. From the statistical analysis (Mann-Whitney unpaired test), the test results between the organic matrix of CHCA and the combi-matrix of CHCA and graphene were independent, with P-values of less than 0.0001. These results showed that the combimatrix of CHCA and graphene could be effectively used for the quantitative analysis of T 4 molecules spiked in serum in a wider concentration range compared to the conventional organic matrix of CHCA.
The clinical laboratories commonly quantify T 4 by RIA, an immunoassay that is complicated to handle and dispose of radioactive materials. In this study, the free T 4 ELISA kit was used as a reference method, and its quantitative analysis results for T 4 were compared to those from MALDI-TOF MS based on the combimatrix of CHCA and graphene. The Bland-Altman plot and Passing-Bablok regression were conducted using MedCalc software (version 18.6) to estimate the statistical coincidence of the two methods for T 4 analysis. The Bland-Altman test revealed that a signal difference is distributed within a confidence level of 95% (± 1.96 σ), as shown in Fig. 8a. This result demonstrated that the two methods were highly correlated and provided similar analysis results for detecting T 4 . In the case of the Passing-Bablok regression, the signals from one method were plotted against the other method when the signals of the two methods had a linear correlation. The analysis data from both methods are distributed at a confidence level of 95%, with a Spearman correlation coefficient (ρ) of 0.973 (p < 0.0001), as shown in Fig. 8b. These results indicated that the two different methods were statistically and highly coincident. Moreover, the two methods were highly correlated and provided similar analysis results for detecting T 4 .

Conclusion
The organic matrix of CHCA was mixed with carbonbased nanostructures of graphene, CNTs, Pt NPs, and P25 for preparing the combi-matrix. The optical properties changed after mixing the solid matrices with CHCA, which were analyzed through UV/VIS spectrometry. The maximum absorption was observed at a wavelength (λ max ) of approximately 260 nm when graphene was mixed with CHCA. The absorption increased at a wavelength of 337 nm of the laser radiation for the ionization of the sample. The results suggested that the absorption of the combi-matrix can achieve a significantly increased absorption of laser radiation for the effective ionization of T 4 molecules. The onset temperature and fusion enthalpy were estimated for analyzing the heat absorption property through DSC. In the case of the combi-matrix, the onset temperature and fusion enthalpy were lower than those of CHCA. These results indicate that the combimatrix can increase the temperature using a smaller amount of heat than CHCA. The analysis of T 4 molecules could be detected with high inter-and intra-spot reproducibility (within 10% RSD) using MALDI-TOF MS based on the combi-matrix of organic matrix and solid matrix. The combi-matrices consisting of CHCA and solid matrices of graphene were used for the quantitative analysis of T 4 molecules spiked in serum after methanol extraction. The standard curve for the analysis of T 4 molecules was prepared from the repeated measurements and the mass peaks, demonstrating high linearity (r 2 < 0.97) at the corresponding concentration range. These results suggest that the combi-matrix consisting of CHCA and graphene can be used for the quantitative analysis of T 4 samples spiked in serum. From the statistical analysis (Mann-Whitney unpaired test), the test results between the organic matrix of CHCA and the combi-matrix of CHCA and graphene were independent with P-values of less than 0.0001. Thus, the combi-matrix of CHCA and graphene can be effectively used for the quantitative analysis of T 4 molecules spiked in serum in a significantly wider concentration range than the conventional organic matrix of CHCA. Finally, the T 4 analysis results with the combimatrix were statistically compared with those of the conventional immunoassay. Fig. 6 Quantitative analysis of T4 in buffer using MALDI-TOF MS based on the combi-matrix. a Spot-to-spot reproducibility. b Shot-to-shot reproducibility. c Standard curves for analyzing T4 using MALDI-TOF MS based on the combi-matrices. d The Mann-Whitney test T4 analysis using MALDI-TOF MS based on the four different kinds of combi-matrices