Hydroxyl-Group-Dominated Graphite Dots Reshape Laser Desorption

Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow .... Alternatively, the surface assisted laser desorption/ionization...
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Hydroxyl-Group-Dominated Graphite Dots Reshape Laser Desorption/Ionization Mass Spectrometry for Small Biomolecular Analysis and Imaging Rui Shi,†,¶ Xing Dai,‡,¶ Weifeng Li,‡,¶ Fang Lu,§ Yang Liu,† Huihua Qu,§ Hao Li,† Qiongyang Chen,∥ He Tian,∥ Enhui Wu,† Yong Wang,⊥ Ruhong Zhou,*,‡,# Shuit-Tong Lee,*,† Yeshayahu Lifshitz,†,∇ Zhenhui Kang,*,† and Jian Liu*,† †

Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices, Institute of Functional Nano and Soft Materials (FUNSOM) and ‡School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, Jiangsu Province 215123, China § School of Basic Medical Sciences, Beijing University of Chinese Medicine, Beijing 100029, China ∥ Center of Electron Microscopy and State Key Laboratory of Silicon Materials, School of Materials Science and Engineering, Zhejiang University, Hangzhou, Zhejiang Province 310027, China ⊥ College of Life Science and Oceanography, Shenzhen Key Laboratory of Marine Bioresources and Ecology, Shenzhen University, Shenzhen, Guangdong Province 518060, China # Department of Chemistry, Columbia University, New York, New York 10027, United States ∇ Department of Materials Science and Engineering, Technion Israel Institute of Technology, Haifa 3200003, Israel S Supporting Information *

ABSTRACT: Small molecules play critical roles in life science, yet their facile detection and imaging in physiological or pathological settings remain a challenge. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) is a powerful tool for molecular analysis. However, conventional organic matrices (CHCA, DHB, etc.) used in assisting analyte ionization suffer from intensive background noise in the mass region below m/z 700, which hinders MALDI MS applications for small-molecule detection. Here, we report that a hydroxyl-groupdominated graphite dot (GD) matrix overcomes limitations of conventional matrices and allows MALDI MS to be used in fast and highthroughput analysis of small biomolecules. GDs exhibit extremely low background noise and ultrahigh sensitivity (with limit of detection 3). (G) MALDI mass spectra of glucose and D-glucose-1-13C in the positive-ion mode. Peak at m/z 203 [glucose + Na]+, peak at m/z 204 [D-glucose-1-13C + Na]+. Inset: Calibration curves for quantitative analysis of glucose. D-glucose-1-13C as the internal standard: 100 pmol.

metabolites) at the suborgan tissue level. We further interpret the high performance of hydroxyl-group-dominated GDs matrix, by revealing the energy-favorable dissociation pathways of carbonfree groups in the negative-ion mode or metal ions (Na+/K+) enrichment and transfer mediated by hydroxyl groups in the positive-ion mode.

properties such as high crystallinity, high aqueous solubility, robust chemical inertness, easy functionalization, and good biocompatibility.41 In previous studies, we have reported strong UV absorption and excellent photochemical properties for GD derivatives.42 Here, we demonstrate that hydroxyl-groupdominated GDs are an ideal MALDI matrix for comprehensive analysis, in situ imaging, and real-time monitoring of small molecules (and their metabolites) in biological samples. They allow for high salt tolerance, fast detection of small molecules with extremely low background noise in the mass range (m/z < 700), and ultrahigh sensitivity (limit of detection (LOD) < 1 fmol, an order of magnitude better than previous reports), including in both the positive-ion and the negative-ion modes. We demonstrate one-step, rapid identification of target small molecules from mixtures of traditional Chinese herb extracts and blood samples without further preseparation. GD matrices enable the direct imaging of small molecules (puerarin and its

RESULTS AND DISCUSSION Synthesis and Characterization of GDs. Pristine GDs were fabricated using an electrochemical etching method in which a high-purity graphite rod was gradually corroded in deionized water.43 This simple and cost-effective manufacturing process can produce highly crystalline GDs with good aqueous dispersity.42 In the present study, we synthesized a series of GDs (Table S1), including the original (i.e., as-prepared) GDs (GD-1) and GD products after reduction (GD-2 to GD-6). The characterization data for GD-4 are presented in the main text due 9502

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either of these two modes. There were only two identifiable peaks indicating Na+ (m/z 23) and K+ (m/z 39) in the positiveion mode using GD-4 as the matrix (Figure 2B).35 A zoomed-in view of the GD-4 mass spectra in the m/z range of 45−800 confirmed its ultraclean background in both the positive-ion and negative-ion modes (Supporting Figure S15). Next, the performance of the GD-4 matrix was tested with glucose and a dipeptide (Ala-Gln) as target biomolecules under the optimized conditions (Supporting Figure S16). The characteristic peaks of glucose such as ([M + Na]+, m/z 203) and ([M + K]+, m/z 219) in the positive-ion mode (Figure 2E), or ([M − H]−, m/z 179) in the negative-ion mode (Supporting Figure S17A) were identified in the MS spectra. The mass spectrum of Ala-Gln in the negative-ion mode displayed the characteristic peak of m/z 216 ([M − H]−) at a high signal-tonoise ratio (Figure 2F). In the positive-ion mode, the characteristic peaks of m/z 240 ([M + Na]+) and 256 ([M + K]+) were clearly detected to identify Ala-Gln (Supporting Figure S17B). These results suggested the high efficiency of GD-4 as the matrix in the LDI of the biomolecules, bypassing the noise or erratic peaks from using organic matrices which had been optimized individually in the gold standard procedure (Supporting Figures S18−21). Specific types of organic matrices may be applied for small molecular analysis by careful selection to avoid undesired overlaps between the ionized analytes and fragmented matrix interference. Peptides were detected by MALDI MS with an empirically chosen matrix (CHCA), although the compounds such as 4-chloro-α-cyano-4-hydroxylcinnamic acid (Cl-CCA) were synthesized to promote detection sensitivity and peptide sequence coverage during analysis, after systematic and targeted screening of the functional groups of the matrix core unit.20,23,48 DHB was reported to be an important matrix choice for the detection of oligosaccharides, while mixture of DHB and N,N-dimethylaniline (DMA, 9) was proposed to improve signal homogeneity, or several other specific organics may be employed to minimize the loss of sialic acid from sialylated glycans by MALDI.49,50 Unfortunately each type of organic matrix still suffers from sporadically distributed noise and limited working windows in the molecular weight range below m/z 700. The mass spectra of glucose or Ala-Gln at the femtomole scale indicated ultrahigh sensitivity assisted by GD-4 matrix for small-molecule analysis (Figure 2E−F and Supporting Figure S17). The LODs of glucose (0.75 fmol, positive-ion mode) and Ala-Gln (0.3 fmol, negative-ion mode) using the GD-4 matrix were further determined by linear extrapolation to signal-to-noise (S/N) = 3 (Supporting Figure S22). These LODs were nearly 3 orders of magnitude better than the traditional organic matrices (CHCA or DHB)50,51 and 1 order of magnitude better than those in previous reports with other carbon nanoparticles as the matrix (10 fmol).35,52,53 The reproducibility of the MS signals was evaluated by testing a microarray of GD-4 matrices. The microarray spots featured a highly homogeneous distribution of the analyte/GD-4 mixture (Supporting Figures S23−24). Thirteen random spot locations were analyzed for MS signal intensities. The coefficient of variation values was lower than 5% in either location-to-location or spot-to-spot homogeneity comparisons, suggesting that highly reproducible MS signals can be harvested using GD-4 matrix. In addition, stable isotope-labeled D-glucose1-13C was employed as the internal standard to quantitatively analyze glucose. Excellent linearity (R2 > 0.999, 0−200 pmol) was obtained between the signal intensity ratios (glucose/ 13 D-glucose-1- C) and glucose concentrations, demonstrating the usefulness of our approach for quantification (Figure 2G).

to its superior performance in MALDI MS. Detailed structural analyses of all samples are available in the Supporting Information. As shown in the transmission electron microscopy (TEM) image (Figure 1A), GD-4 particles were well dispersed and approximately 5−6 nm in size. A high-resolution TEM image (Figure 1B) of a GD-4 particle exhibited a well-crystallized graphitic honeycomb structure. Selected-area electron diffraction (SAED) was performed on this region along the {002} zone axis, highlighting the hexagonal single-crystal structure of the graphitic sheets (Figure 1C). The highly graphitic nature of GD-4 was further verified by analyzing a high-resolution TEM image of another GD on the {121} zone axis (Supporting Figure S1).44 A tapping-mode AFM imaging (Figure 1D) revealed that the GD-4 height was consistent with the lateral dimension (approximately 6 nm). Good dispersity for GD-4 in aqueous solution was demonstrated by dynamic light scattering (DLS) (median hydrodynamic size 14 nm, Figure 1E). In the Raman spectrum of GD-4 (Figure 1F), the peak at 1350 cm−1 (D band) was attributed to the vibration of sp3-bonded carbon atoms in disordered graphite, while the peak at 1600 cm−1 (G band) corresponding to the vibration of sp2-bonded carbon atoms in a two-dimensional (2D) hexagonal lattice.45 GD-4 exhibited relatively strong absorption in the UV−vis wavelength range (Supporting Figures S2−3), either in the solution or in the solid phase. The most widely used wavelengths of laser for MALDI MS include 337 and 355 nm. This finding suggests that GD-4 was a good receiver of laser energy for the subsequent ionization process.46 The detailed characterization data from IR spectrometry (Supporting Figure S4), X-ray photoelectron spectroscopy (XPS) (Supporting Figure S5), and elemental analysis (Table S1) revealed oxygen-containing groups on the surface of GD-4 and the high purity of GDs free of metal contaminants. The hydroxyl (1.4 × 10−19 mol/particle), carbonyl (0.7 × 10−19 mol/particle), and epoxy (0.05 × 10−19 mol/particle) groups on the surface of GD-4 were further quantified by conductivity titration (Supporting Figure S6). The characterizations (TEM, AFM, IR, XPS, UV−vis, Raman, and elemental analysis) of other GDs samples in the series are presented in Supporting Figures S7−12 and Table S2. A major difference between GD-4 and the pristine GD-1 was a significant 2-fold increase in hydroxyl groups and a decrease in carbonyl groups (by 30%) and epoxy groups (7-fold) on the surface. These oxygen-containing groups are important for the dispersity of GDs in aqueous solutions (Supporting Figure S13). However, excessively reduced GDs (such as GD-5 and GD-6 in Supporting Figure S13) had lower dispersity in aqueous solutions, which would compromise the performance of these GDs as a MALDI matrix in terms of signal reproducibility.47 Low-Noise and High-Efficiency of GD-4 Matrix for Small Biomolecular Analysis. The mass spectra of GD-4 were compared against traditional matrix molecules (CHCA: α-cyano4-hydroxycinnamic acid, SA: sinapic acid, DHB: 2,5-dihydroxybenzoic acid) under an identical laser pulse energy (57 μJ), including both the positive-ion and the negative-ion modes. As shown in Figure 2A−D, extremely intense noise bumps from fragments of the traditional matrix molecule (CHCA) appeared in the range of m/z 0−800. Additional mass spectra are presented in Supporting Figure S14 as a reference, including other organic matrix molecules (DHB and SA) in either the negative-ion or positive-ion mode. All organic matrices suffered from a very noisy and intrinsic background signal below m/z 800, which was generated during the laser desorption ionization (LDI) process.40 In the sharp contrast, the mass spectra of GD-4 featured a clean background, free of noise interference from the matrix itself in 9503

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Figure 3. GD-4-assisted MALDI MS for identification of oligosaccharides from a Chinese herb extract and analysis/imaging of small molecular metabolites in serum or tissue. (A) MALDI mass spectra of Morinda officinalis extract in positive-ion mode. DP2: sucrose, DP3: trisaccharide, DP4: tetrasaccharide, DP5: pentasaccharide, DP6: hexasaccharide, DP7: heptasaccharide, DP8: octasaccharide, DP9: nonasaccharide. Inset: zoomed-in view of the spectrum in m/z 1300−1600. (B) Tandem MS spectra of oligosaccharides (DP8 and DP9). (C) MALDI mass spectrum (negative-ion mode) of mouse serum (1 μL) 30 min after intraperitoneal administration of puerarin (dosage: 500 mg/kg). Inset: Zoomed-in view of the spectrum in m/z 410−430, characteristic peak of puerarin: [M − H] at m/z 416. (D) Metabolic pathways showing puerarin and its two metabolites (daidzein and dihydrodaidzein). (E) MALDI MS images of the drug and its metabolites (puerarin, m/z: 416; daidzein, m/z: 253; and dihydrodaidzein, m/z: 255) in a kidney tissue slice with an optical micrograph of an H&E-stained consecutive slice as a reference. The color bar encodes the signal intensities of the three small molecules in MSI. Scale bar: 2000 μm.

unit (Supporting Figure S27).56 The direct analysis of long chained natural oligosaccharides such as Morinda off icinalis extract by MALDI MS using conventional matrices remains a challenge49,57−60 (Supporting Figures S28−29). However, we have demonstrated that GD-4 is an effective matrix to analyze the oligosaccharides in the Morinda off icinalis extract. As shown in Figure 3A, the presence of a whole family of inulin-type oligosaccharides (DP2−DP9) from the Morinda off icinalis extract was revealed by a single MALDI MS spectrum. For each member of the inulin-type oligosaccharide family, a detailed analysis became available via tandem mass (MS/MS) spectra due to the high quality of the GD-4 matrix signal. The tandem mass spectrometry data for DP8 (m/z 1353) and DP9 (m/z 1499) are displayed in Figure 3B. A series of periodic peaks in the tandem mass spectra corresponded to fragmented oligosaccharides with an interval (m/z = 162) due to glycosidic cleavages. The data represent the successful analysis of the most challenging

The performance of the GD-4 matrix was further investigated using a family of oligosaccharides as target analytes. The critical role of oligosaccharides is well acknowledged in mediating cell−cell recognition and cell adhesion.54 However, the oligosaccharides are difficult to analyze using conventional organic MALDI matrices (Supporting Figure S25). Note that the GD-4 matrix enabled fast and sensitive detection of oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose) by MALDI MS (Supporting Figures S25−26). It did not require the tedious desalting steps, as a dramatic advantage of the procedure simplification for oligosaccharide analysis. Morinda off icinalis is a traditional Chinese herb with a clinical record of antidepressant activities.55 Its extract contains a mixture of inulin-type oligosaccharides that are composed of fructose unit chains in various degrees of polymerization (DPn, where n is the number of fructose units) and are terminated by a single glucose 9504

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Figure 4. Mechanistic basis of hydroxyl-group-dominated GD matrix by experiments and DFT simulations. (A) Heat map of the MALDI MS background noise intensity relative to the content of oxygen-containing (epoxy and carbonyl) groups and laser pulse energy. (B) Heat map of the MALD IMS signal-to-noise ratio relative to the content of hydroxyl groups and laser energy. Analyte: Ala-Gln. Negative-ion mode. (C) C 1s spectra of the mixture (GD-4 and n-dodecanethiol) before and after laser (355 nm) irradiation under a nitrogen atmosphere. (D) Dissociative pathways of different oxygen-containing (epoxy, carbonyl, and hydroxyl) groups from GDs by DFT simulations. Dissociation energies (ΔE) were calculated at B3LYP-D3/6-31G(d, p) level. The energy-favorable pathways are distinguished from the others by using the blue color-encoded symbols. Enlarged carbon atoms are used to highlight the local defects on the surface carbon sheet. The gray, red, and white balls express the carbon, oxygen, and hydrogen atoms, respectively.

neurological disorders.62,63 Mouse sera were collected in a time series (20 min) after intraperitoneal administration of puerarin. The characteristic peak of puerarin was clearly detected in the MS spectra ([M − H]−, m/z 416, negative-ion mode) (Figure 3C and Supporting Figure S31A−B) and further verified by tandem MS (Supporting Figure S31C−D). The time-course profiles indicated that puerarin was quickly absorbed into the blood, and the maximum concentration was achieved within 25−30 min of intraperitoneal administration (Supporting Figure S31E). Retention of puerarin in the blood was reduced to a minimal level after 12 h.64 Daidzein and dihydrodaidzein, two important metabolites of puerarin (Figure 3D),65 were also identified by characteristic peaks at m/z 277 and 279, respectively (Supporting Figures S32−33). Our results simultaneously revealed the pharmacokinetic behavior of daidzein and dihydrodaidzein in response to the change in puerarin (Supporting Figures S32E and S33E), consistent with the previous reports.66 For the purpose of comparison, optimization of the traditional organic matrices (CHCA or DHB) was performed in parallel to determine the best experimental condition to detect puerarin (Supporting Figures S34−37). But the MALDI MS spectra under the optimal conditions for CHCA or DHB still suffered from the high-noise

oligosaccharide components in MS. A detailed analysis of the other oligosaccharide members in the tandem mass spectra is available in Supporting Figure S30. The oligosaccharides of DP2−DP9 in the Morinda of f icinalis extract were also analyzed by the conventional HPLC technique for a side-by-side comparison. Notably, the oligosaccharides DP8 and DP9 in the Morinda of f icinalis extract were below the detection limit of HPLC. However, the GD-4-assisted MALDI MS technique enabled the acquisition of sizable signals for DP8 and DP9 directly from the Morinda off icinalis extract, due to its ultrahigh sensitivity and the elimination of long, tedious sample preparation. Rapid Analysis and in Situ Imaging of Small Biomolecules/Metabolites. Therapeutic drug monitoring in serum is an essential workflow for pharmadynamic studies, which requires high throughput and sufficient sensitivity. N-doped graphene was used as the matrix to detect anticancer drugs spiked in serum by MALDI MS.61 Here we demonstrate that the GD-4 matrix enabled the rapid analysis of target small molecules and their metabolites in blood serum or sectioned tissue samples without any need of purification. Puerarin is a major bioactive isoflavone (daidzein-8-C-gluocoside, MW: 417) extracted from the root of Pueraria lobata for the treatment of cardiovascular or 9505

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by our control experiments (Supporting Figure S39).74 Herein, our GDs provide the ultraclean and broad-spectrum matrix for directly mapping small molecules and their metabolites simultaneously at suborgan tissue level by MSI, which may reshape this technology for small molecular analysis of metabolomics and tissue-level multiplexed molecular imaging. Mechanistic Basis of Hydroxyl-Group-Dominated GD Matrix. We attempted to investigate the mechanistic basis of the high-performance hydroxyl-group-dominated GD matrix by a combination of experiments and density functional theory (DFT) simulations. In Figure 4A, a heat map is presented for the background noise (measured in the negative-ion mode) levels related to the percentages of carbonyl or epoxy groups on GDs and laser pulse energy. The GD series exhibit distinct patterns in terms of the background noise under the laser pulse energy titration. There exists significant positive correlation between the carbonyl or epoxy group removal and the reduced background noise intensity (GD-1 to GD-4). However, a rebound of the background noise results from excessive removal of GD hydroxyl groups (GD-5 and GD-6), likely due to an increasing risk of self-decomposition by laser ablation after over-reduction.75 In Figure 4B, the relationship between the hydroxyl group content and the S/N ratios was plotted under a wide range of laser pulse energy from 49 to 69 μJ. GDs containing a higher content of hydroxyl groups on the surface would allow for the detection of small molecules with the improved S/N ratios. Among them, GD-4 with the highest percentage of hydroxyl groups performed best as the matrix for MALDI MS and required only a low laser pulse energy (∼50 s μJ) for the LDI process, which was a great advantage in reducing noise and prolonging laser lifetime. Due to the variable effect of loss of the laser power on optical elements within the ion source, it is difficult to directly compare the input laser energy (or power) between different laboratories or against the reported values in the literature. Careful calibration of the laser intensity of the ion source and application of standard instrumental parameters are necessary to interpret the comparability of laser energy levels.36,76−78 In addition, the consumption of hydroxyl groups (on GD-4) was validated by investigation on O element changes of GD-4 mixed with a carefully selected analyte under the laser irradiation. The mixture of GD-4 and n-dodecanethiol was dropped in a piece of indium and dried at room temperature. The sample was irradiated by UV pulse laser (355 nm) for 30 s under nitrogen atmosphere. Identical samples with or without laser eradication were characterized by the XPS to compare the change of the surface groups. As shown in Figure 4C, the hydroxyl group content (O 1s spectral analysis) of GDs was reduced from 25% to 11%, which suggested a significant consumption of hydroxyl groups during the reaction induced by the laser irradiation. Quantum mechanical simulations based on DFT were performed for a deep understanding of GD-assisted MALDI MS. As shown in Figure 4D, a hexagonal graphene sheet (containing 19 six-member rings) was used to mimic the GD surface. The dangling carbons were saturated by H atoms. Four representative scenarios, including surface epoxy group, surface hydroxyl group, edge carbonyl group, and edge hydroxyl group, were considered in our DFT calculations. For each scenario, two potential pathways of dissociation were investigated. The geometries of each reactant and product were fully optimized at B3LYP-D3/6-31G (d, p) level. The dissociation energies, ΔE, were calculated by the formula of ΔE = E(product) − E(reactant). When carbonyl or epoxy groups were predominant on the GDs, they tended to be involved with the dissociative pathways of generating carbon

level which overwhelmingly suppressed the puerarin signal in mouse serum (m/z 416 undetectable). The standard procedure for metabolic analysis requires many steps, including preseparation of small molecules by liquid chromatography (LC) (Supporting Figure S38) or multiple reaction monitoring (MRM) techniques for a cascade of filtration.65 In contrast, our approach enables bypassing the tedious LC and MRM procedures, allowing a direct analysis of small molecules and their metabolites from fresh biological samples with minimal processing.64 This process not only produced data faster but also reduced the sample volume to as low as 1 μL (several orders of magnitude lower than LC/MRM).64 The significant savings in sample volume would alleviate the burden for blood sampling and facilitate the technical development of large-scale screening for small-molecule drugs and their metabolites.64 We further demonstrated the high-resolution mapping of small molecules and their metabolites in tissue sections by GD-4-assisted mass spectrometric imaging (MSI). MALDI MSI is a label-free and high-throughput (multiplexed analysis of hundreds to thousands of molecules) imaging technique to correlate their molecular information with traditional histology. Organic matrices assist MSI to achieve great success in reconstructing the pathological distribution of proteins or higher mass molecules (>m/z 2000 typically). Some small molecular pharmaceuticals, vitamins, and metabolites have also been imaged in different types of tissues with various organic matrices. Phospholipids in the m/z range of 400−900 are deeply investigated by MS imaging due to relatively little interference with matrix peaks.67−70 Nevertheless, organic matrices are still limited for small molecular imaging by the barrier: each specific organic matrix requires a careful preselection and provides only a few occasional, narrow m/z windows surrounded by the matrix fragmentation noise, thus being difficult to visualize different groups of analytes simultaneously.71−74 The spatial resolution of MS imaging is also influenced by the various sizes of organic matrix crystals and analyte extraction/migration across the tissue specimen by organics.49 Our GD matrix addressed this challenge with outstanding advantages. Distribution of puerarin and its metabolites daidzein and dihydrodaidzein was located in the kidney tissue slice based on their characteristic MALDI MS signals (Figure 3E). The consecutive tissue slice was stained by H&E staining as the reference. For each small-molecule analyte, the signal intensities of the characteristic peaks in the mass spectra were symbolized by different colors in the heat map. There were distinct patterns for the relative abundance of puerarin, daidzein, and dihydrodaidzein in the kidney tissue slice. Puerarin (m/z 416) was primarily distributed in the renal pelvis and major calyx. It was also present in the renal parenchyma (cortex, medulla) at a relatively lower abundance than that at other sites due to the filtration effect in the glomerular capillary wall. Daidzein (m/z 253) and dihydrodaidzein (m/z 255) were confined to tissue areas such as the renal pelvis and major calyx and partly in the minor calyx as well. Interestingly, these metabolites (daidzein and dihydrodaidzein) were also distributed near the renal cortex, but were nearly absent in the medulla. The metabolism of puerarin was involved in a cascade of reactions, including cleavage of the glucose unit and a subsequent reduction of daidzein to dihydrodaidzein (Figure 3D). Different distribution patterns indicated metabolism by separate steps and discriminable retention of small molecules in suborgan tissues. In the literature, imaging/mapping of small molecules (m/z < 700) in tissue specimens was very challenging because of the interference of complicated organic matrix fragments, which was also confirmed 9506

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Figure 5. Geometry optimization of the analyte, energy calculations, and schematic drawing of GD-4-assisted ionization of small molecules in the negative-ion mode. (A) The optimized geometry structure of the dipeptide Ala-Gln molecule, showing three candidate sites (dotted circles) for deprotonation indicated by the corresponding energy requirements. The gray, red, blue, and white balls express the carbon, oxygen, nitrogen, and hydrogen atoms, respectively. (B) Electrostatic potential (ESP) on the van der Waals surface (electron isodensity = 0.001 au) of Ala-Gln. (C−D) Energy landscape for reactions involving the analyte (R-COOH) and the dissociative oxygen-containing groups of GDs in the negativeion mode. (C) An OH radical dissociated from the GDs reacts with the analyte (R-COOH). (D) An OH− anion dissociated from the GDs reacts with the analyte (R-COOH). The curves represent the relaxed potential energy surface for the H atom or the proton-transfer process. The gray, white, and red balls indicate the C, H, and O atoms. (E) Mechanism of hydroxyl-group-dominated GDs as a matrix promoting the ionization of small molecules (H−M).

fragments and secondary structural defects with the risk of higher background noise. In contrast, the hydroxyl-group-dominated GDs were able to find energy-favorable pathways (0.48 eV vs 9.93 eV; 4.98 eV vs 6.90 eV) for the dissociation of carbon-free groups, thus dramatically reducing background noise (Figure 4D). Essentially, the hydroxyl-group-dominated GDs are featured with enhanced structural stability under laser irradiation, by sacrificing the hydroxyl groups through the energy-favorable dissociative pathways to minimize the local defects on GD structure. The dipeptide of Ala-Gln was selected as the representative analyte in the negative-ion mode of MALDI MS, using a fully optimized geometry structure at B3LYP-D3/6-31G(d, p) level (Figure 5A). The energy requirements for deprotonations at three terminals (−COOH, −CONH2, and −NH2) were, respectively, calculated, while the total energy of the ground state of the neutral Ala-Gln molecule was defined as zero, and then the electrostatic potential (ESP) was derived based on the ground state electron density (Figure 5B). These three hydrogenous

terminals could potentially be adsorbed onto GD surface, interacting with the oxygen-containing groups of the GDs through hydrogen bonding. However, given the effect of the ESP maximum on the electron density surface near the carboxyl terminal (−COOH), it would be most conducive for the carboxyl terminal to approach the chemical groups of GDs. In addition, the carboxyl terminal requires the smallest energy input for deprotonation in our calculation. Therefore, a reasonable preassumption was made to focus on the proton transfer that originated from the carboxyl terminal (−COOH) of Ala-Gln in the negative-ion mode of MALDI MS, using a further simplified molecular model (R-COOH). The carbon-free, oxygen-containing groups dissociated from the GDs by laser irradiation can be either radicals (OH•) or anions (OH−) under the complex environment of laser photochemistry. Both cases were considered in our DFT calculations to investigate how these oxygencontaining groups can react with analyte (R-COOH) for the H atom or the proton-transfer process. As for the OH• radical, 9507

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Figure 6. Theoretical prediction and experimental verification of small molecules containing characteristic chemical groups by GD4-assisted MALDI MS. (A) Theoretically calculated Gibbs free energy change (ΔG) of a hypothetical proton transfer reaction, M + OH− →M− + H2O, for seven representative hydrogenous molecular terminals (M). Each molecular terminal was saturated by a methyl, as shown in the ball-and-stick models. Each reactant and product in the hypothetical reaction were optimized at MP2/6-311++G(d, p) level. (B) The chemical formulas of seven selected analytes closest to these representative molecular terminals. (C) GD4-assisted MALDI MS spectra of the selected analyte panel as experimental verification of theoretical calculations in the negative-ion mode.

(OH− or OH•). Lastly, the carbon-free hydroxyl species rapidly capture protons from the analytes through energy-favorable pathways, generating negatively charged small-molecule fragments for MALDI MS analysis. Furthermore, ab initio calculations based on the above model predicted the thermodynamic feasibility for detecting various types of small molecules in the negative-ion mode. A panel of representative molecules containing different chemical groups was selected for ab initio calculations of the Gibbs free energy changes (Figure 6A). The results indicated that five types of small molecules (R-SO3H, R-COOH, R-SH, R-CONH2, R−OH) could accomplish proton transfer (ΔG < 0) with the dissociative hydroxyl groups, while the process would be thermodynamically difficult to complete with the other types (R-CHO, R-CH3) (ΔG > 0). A panel of seven analytes closest to these representative molecular terminals was selected for MALDI MS spectra to test ab initio calculations (Figure 6B). The theoretical prediction was confirmed by MALDI MS measurements using the GD-4 matrix (Figure 6C and Supporting Figure S40). Therefore, our mechanistic model by DFT can offer very useful information to guide the practical measurements in GD-4-assisted MALDI MS.

the calculated energy barrier of hydrogen transfer is very small, 0.1 eV approximately, in the calculation of relaxed reaction potential surfaces (Figure 5C). On the other hand, the proton transfer for the OH− anions is a spontaneous reaction with no energy barrier (Figure 5D). Therefore, either case (the low energy barrier (0.1 eV) or no energy barrier) indicates that these dissociative oxygen-containing groups are highly reactive, promising energy-favorable mechanisms in the negative-ion mode. Our DFT calculations support the high sensitivity in the small molecular detection using the hydroxyl-group-dominated GDs as matrices for MALDI MS. As a summary of our experimental results and DFT calculations, we propose a mechanism of hydroxyl-group-dominated GDs as the MALDI matrix for small molecules, as illustrated in Figure 5E (in the negative-ion mode). Small molecular analytes tend to be adsorbed onto the GD surface through hydrogen bonding and van der Waals interactions. The subsequent ionization process benefits mostly from the hydrogen-bonding interactions between the hydroxyl group of GD-4 and the hydrogen atom of the analyte. The input laser energy can efficiently be transduced by GD-4, initiating the dissociative process of carbon-free hydroxyl species 9508

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Mettler-Toledo TGA/DSC. The size distribution of GDs in aqueous solution was analyzed by a DLS instrument (Zetasizer Nano ZS, ZEN 3690, Malvern). Synthesis of GDs. Pristine GD-1 was synthesized using an electrochemical etching method. Briefly, two parallel graphite rods (99.99%, Alfa Aesar Co. Ltd.) were used as the anode and cathode in deionized water. A static potential of 30 V was applied to the two electrodes under continuous intense magnetic stirring to gradually produce GD-1 in the solution (dark-yellow color). The obtained solution was filtered and then centrifuged at 12,000 rpm for 30 min to remove the precipitated graphite oxide and graphite particles. The supernatant was centrifuged at 22,000 rpm for 10 min to collect the nearly monodisperse GD-1 (approximately 5−6 nm). The other GDs (GD-2 to GD-6) were synthesized by reducing the pristine GD-1 under a range of conditions. Briefly, 100 mg of GD-1 was mixed with a solution of 100 mL of NaBH4 (50 mM for GD-2; 100 mM for GD-3; 150 mM for GD-4). The mixture was stirred at room temperature for 10 h and purified with a dialysis membrane (3500 Da) against deionized water to remove excess salt. To prepare GD-5 and GD-6, 100 mg of GD-4 was dispersed in 100 mL of deionized water, followed by the addition of hydrazine hydrate (0.2 mL for GD-5; 1 mL for GD-6). The mixture solution was refluxed under magnetic stirring (at 60 °C for GD-5; at 80 °C for GD-6) for 6 h. The solution of GD-5 or GD-6 was purified using a dialysis membrane (3500 Da) against deionized water to remove excess salt. Quantification of Oxygen-Containing Groups on GDs. Conductometric titration was performed to estimate the percentage of oxygen-containing groups on GDs. A conductance titrator (DDS-11A) was used in the experiments. GDs (0.1 mg mL−1, 100 mg), 40 mL of solvent (pyridine/acetone = 1/4), 1 mL of distilled water, and 1 mL of ethanol were mixed in a 50 mL four-mouth flask. The mixture was stirred at 150 rpm at 25 °C for 10 min. A solution of 0.05 mol L−1 KOH in isopropyl alcohol was added to the mixture for titration, and the conductometric signals were simultaneously recorded. Typically, the equivalence points in the conductometric curve (Supporting Figure S6) were determined by the intersection of the tangential lines, using point A or B for the carbonyl or hydroxyl groups, respectively. The molarities of the carbonyl or hydroxyl groups were calculated using the following formulas:

Our calculations also provided a good theoretical explanation for the positive-ion mode using GD-4 matrix. The α-D-glucose was chosen as the simplified molecular model of sugar family. Our DFT calculations validated strong coordinate interactions between single Na+ cation and multiple O atoms of the sugar molecule in the binding energy range from −40 to −60 kcal/mol approximately (Supporting Figure S41). It explained the high possibility of detecting the Na+/glucose signal in the positive-ion mode of MALDI MS. The detailed potential energy surface simulations at a semiempirical PM3 level suggest a dislocation of the glucose following the movement of the Na+, thus providing a dynamic view for the dissociative mechanism of Na+/glucose from GDs in the positive ion mode (Supporting Figure S42).

CONCLUSIONS In summary, we have developed hydroxyl-group-dominated GDs as an ideal MALDI matrix for small molecular analysis and in situ imaging in biological samples. They break the barriers faced by traditional organic matrices in the detection of molecules (m/z < 700), exhibiting extremely low background noise and ultrahigh sensitivity. Our experimental results and theoretical calculations suggest that the properties of GDs and the species of chemical groups on the surface (e.g., hydroxyl groups) are critical for the high performance in MALDI MS applications. Proteins or polymers of higher mass can be ionized by GD-4 matrix, and in their mass spectra, we observed relatively broad peaks for these macromolecules with multiple charges in some typical cases (Supporting Figure S43). The phenomenon is interesting, but also requires further investigation in the future. This current work suggests that the applications of GD matrix should only be focused on small molecules. Indeed, varieties of small biomolecules have been tested by GD-assisted MALDI MS with a great success. Hydroxyl-group-dominated GDs exhibit high quality and a long shelf life. After being stored at room temperature for more than 2 years, they maintain the original dispersity in aqueous solutions and high performance in the MALDI MS detection of varieties of small molecules (Supporting Figure S44). A significant saving in laser energy consumption by our GD matrix is favorable for not only laser lifetime but also improved spatial resolution in MALDI MS imaging. Our findings will trigger tremendous efforts in this field by creating an expressway for MALDI MS to analyze and directly visualize small-molecule analytes in biological samples.

n(carbonyl) = VAC

n(hydroxyl) = (VB − VA )C MALDI MS Experiments with GDs and Conventional Matrix. MALDI TOF MS analysis was performed on a Bruker BIFLEX III mass spectrometer (Bruker Daltonics, Germany) equipped with an Nd:YAG laser (wavelength 355 nm, laser pulse duration 3 ns) with reflection in the positive-ion and negative-ion modes. The laser energy was adjusted between 0% and 100%, corresponding to the scales of 45 μJ and 85 μJ per pulse, respectively. A two-layer method was mainly applied to prepare the spots of the GD matrix/analytes. The GD solution (1 μL, 1 mg/mL) was dropped onto the MALDI sample plate and dried at room temperature. The analyte solution (1 μL) at the specified concentration was deposited on the GD surface and dried at room temperature prior to any MALDI MS tests. The traditional organic matrices (CHCA or DHB) were applied in a standard method. The saturated CHCA solution (ACN 30%, TFA 0.1%, v/v) or DHB matrix solution (10 mg/mL, ACN 30%, TFA 0.1%, v/v) was first prepared, stored at 4 °C and used as needed. One μL CHCA or DHB solution was deposited on the steel plate and dried at room temperature. Then 1 μL of the sample mixture of the analytes and organic matrices in the specified ratios was added on the plate and then dried at room temperature. The MALDI TOF MS experiments were performed with the positive-ion and negative-ion modes. Preparation and Analysis of Morinda of ficinalis Extract. The dried roots of Morinda of ficinalis F. C. How were purchased from the Beijing Tongrentang Group Co., Ltd., China. The plant was authenticated by the Chinese Medicine expert Y. Zhao of the Beijing University of Chinese Medicine. The roots were ground to a fine powder and extracted with water (90 °C) in a 1:20 (w/w) ratio for 3 h. After the

MATERIALS AND METHODS Chemicals. All chemicals were purchased from Sigma-Aldrich, Adamas Bate, or Beijing Chemical Reagent (Beijing, China) and used as received without further purification. Deionized water (18 MΩ) from Milli-Q was used in all experiments. Characterization. Transmission electron microscopy (TEM) images were obtained with an FEI/Philips Tecnai G2 F20 TWIN TEM. The Fourier transform infrared (FT-IR) spectra of GDs were obtained with a Bruker FT-IR spectrometer (Hyperion). Raman spectra were collected using a high-resolution 800 Raman spectroscope (J Y, France). UV−vis spectra were obtained with a PerkinElmer UV−vis spectrophotometer (lambda 750). X-ray photoelectron spectra (XPS) were measured using a KRATOS Axis Ultra DLD X-ray photoelectron spectroscope. MALDI TOF MS experiments were performed on a Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Inc., Billarica, MA) with an Nd:YAG laser (355 nm, the laser spot size of 50−100 μm, 2000 kHz), using a software package of Bruker DataAnalysis 3.3. Atomic force microscopy (AFM) measurements were performed on a Veeco Multimode V atomic force microscope. The thermogravimetric analyses (TGA) were conducted on a 9509

DOI: 10.1021/acsnano.7b05328 ACS Nano 2017, 11, 9500−9513

Article

ACS Nano *E-mail: [email protected].

extract solution was cooled to room temperature, 4-fold volumes of 95% ethanol were added to form a mixture for precipitation at 2 °C overnight. The precipitate was filtered using filter paper. The transparent extracting solution was collected and evaporated on a rotatory evaporator under reduced pressure. The final concentration was approximately 10 mg/mL in terms of dried plant weight in the aqueous extract. The solution was diluted 1000-fold before MADLI MS testing. Animal Experiments and Drug Administration. Kunming mice weighing 25 ± 3 g were obtained from Vital River Laboratories (Beijing China). Mice had free access to animal food (Keaoxieli Animal Feed Co., Ltd., Beijing, China) and water, which were replenished twice a week. All animal care and experimental procedures were conducted according to the Guidelines for Animal Experiments of the School of Basic Medical Sciences, which were approved by the Committee on Ethics of Animal Experiments at Beijing University of Chinese Medicine, China. The mice (n = 72, divided into 6 groups) received an intraperitoneal administration of a single dose of 0.5 g/kg of puerarin dissolved in 0.05 M CBS buffer solution (pH 9.6). Blood samples (1 mL) were obtained at 10 min, 20 min, 30 min, 45 min, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after drug administration. An additional six mice were sacrificed predose to provide control serum for analysis. One h after blood sampling, 1.2 mL of methanol was added to 300 μL serum samples (4:1, v/v) in a 5 mL glass centrifuge tube for rapid mixing. The serum samples were separated by centrifugation (3000 rpm, 15 min) to collect the supernatant. Preparation of Tissue Slices. The dissected organs from the sacrificed mice were flash frozen by slow immersion in liquid nitrogen for 10 min and stored in a refrigerator at −20 °C. The frozen tissues were sectioned at −20 °C into 10 μm-thick slices in a Leica CM-1950 cryostat (Leica Biosystems). The tissue slices for MALDI MS imaging or immunohistochemistry (IHC) staining were prepared from the mouse kidney. GD-4 (1 mg/mL) was dropped on indium tin oxide-coated glass slices (Bruker Daltonics) and dried at room temperature to form a uniform thin film. The tissue sections were thaw mounted on the surface of GD-4 and dried under vacuum at a pressure of 10 mbar for 30 min. They were imaged by a Bruker Ultraflextreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Inc., Billarica, MA) with an Nd:YAG laser (355 nm) (accumulation: 2000 shots; laser spot size: 50−100 μ, pixel size: 100 μ) in the negative-ion mode. The laser pulse energy was set to be on the level of 30% (57 μJ). Bruker FlexImaging software (version 4.0) was used for MSI and tissue image reconstruction. The adjacent kidney slices were also stained using a standard IHC procedure, followed by optical scanning with a CanoScan 9000-F scanner (Canon) for a reference. Computational Details. All calculations in this work were performed using Gaussian 09 program.79 The computational models and methods are detailed in the corresponding sections of Supporting Information.

ORCID

Weifeng Li: 0000-0002-0244-2908 Ruhong Zhou: 0000-0001-8624-5591 Shuit-Tong Lee: 0000-0003-1238-9802 Zhenhui Kang: 0000-0001-6989-5840 Jian Liu: 0000-0002-0095-8978 Author Contributions ¶

These authors contributed equally to this work. J.L. and Z.H.K. conceived and designed the experiments. R.S. synthesized GDs and performed the MS experiments. X.D. and W.F.L. performed the theoretical calculations and participated in the writing. F.L., H.H.Q., and E.H.W. assisted with the animal experiments. Y.L. and H.L. helped prepare the GDs. Q.Y.C. and H.T. provided help in TEM analysis. Y.W. and E.H.W. assisted with MS measurements. R.S., J.L., Z.H.K., R.H.Z., and S.T.L. wrote the manuscript. Y. Lifshitz participated in the writing discussions.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work is supported by the National Science and Technology Major Project Fund (2011ZX02707), the National Basic Research Program of China (973 Program) (2013CB932702), the National Natural Science Foundation of China (21275106, 51422207, 21575095, 11374221, 51132006, 51572179, 21471106, 21501126, 11574224, and 21320102003), the Specialized Research Fund for the Doctoral Program of Higher Education (20123201110018), Collaborative Innovation Center of Suzhou Nano Science and Technology, and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). X.D. is supported by China Postdoctoral Science Foundation (2016M591901). J.L. is supported by the “1000 Youth Talents” plan of the Global Expert Recruitment Program. We thank Dr. Jinjuan Xue, Prof. Zongxiu Nie, and Prof. Weiguo Song from the Institute of Chemistry, Chinese Academy of Sciences and Prof. Guoqiang Xu from Soochow University for profound discussions. REFERENCES (1) May, M. Synthetic Biology’s Clinical Applications. Science 2015, 349, 1564−1566. (2) Wolan, D. W.; Zorn, J. A.; Gray, D. C.; Wells, J. A. Small-Molecule Activators of a Proenzyme. Science 2009, 326, 853−858. (3) Stockwell, B. R. Exploring Biology with Small Organic Molecules. Nature 2004, 432, 846−854. (4) Scott, D. E.; Bayly, A. R.; Abell, C.; Skidmore, J. Small Molecules, Big Targets: Drug Discovery Faces the Protein-Protein Interaction Challenge. Nat. Rev. Drug Discovery 2016, 15, 533−550. (5) Biggar, K. K.; Li, S. S. Non-Histone Protein Methylation as a Regulator of Cellular Signalling and Function. Nat. Rev. Mol. Cell Biol. 2015, 16, 5−17. (6) Evans, A. M.; DeHaven, C. D.; Barrett, T.; Mitchell, M.; Milgram, E. Integrated, Nontargeted Ultrahigh Performance Liquid Chromatography/Electrospray Ionization Tandem Mass Spectrometry Platform for the Identification and Relative Quantification of the Small-Molecule Complement of Biological Systems. Anal. Chem. 2009, 81, 6656−6667. (7) Steppan, C. M.; Brown, E. J.; Wright, C. M.; Bhat, S.; Banerjee, R. R.; Dai, C. Y.; Enders, G. H.; Silberg, D. G.; Wen, X.; Wu, G. D. A Family of Tissue-Specific Resistin-Like Molecules. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 502−506. (8) Bleicher, K. H.; Bohm, H. J.; Muller, K.; Alanine, A. I. Hit and Lead Generation: Beyond High-Throughput Screening. Nat. Rev. Drug Discovery 2003, 2, 369−378.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b05328. Details of characterization of GDs including TEM and AFM images, XPS and Raman spectrum, and other techniques. Mass spectra of dipeptide, glucose, and oligosaccharides family using conventional matrices and GD4. Mass spectra of Morinda off icinalis and puerarin using CHCA, DHB, and GD4 as the matrix. Mechanistic basis of hydroxyl-group-dominated GD matrix. Tables of the element analysis and functional groups on GDs (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. 9510

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DOI: 10.1021/acsnano.7b05328 ACS Nano 2017, 11, 9500−9513

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ACS Nano B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian 09, Revision E.01; Gaussian, Inc.: Wallingford, CT, 2013.

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DOI: 10.1021/acsnano.7b05328 ACS Nano 2017, 11, 9500−9513