Rapid and sensitive serum glucose determination using chemical labeling coupled with black phosphorus-assisted laser desorption/ionization time-of-flight mass spectrometry
Graphical abstract
Introduction
Glucose is a hexose that serves as the primary energy source for living cells in the human body, and its concentration levels in serum are widely recognized as a critical clinical indicator of diabetes mellitus [1]. Over time, the presence of too much glucose in the blood can lead to severe secondary complications, such as retinopathy, nephropathy, neuropathy, and cardiovascular disease [2]. In addition, emerging research has shown that the serum glucose level is closely related to pancreatic, esophagus, liver, and colon/rectum cancers [3], indicating that serum glucose may be a potential biomarker for monitoring the development of cancer. Therefore, numerous methods have been investigated for blood glucose detection. In recent years, electrochemically or optically developed glucose sensors [4], [5] are commonly used due to their low-cost and portability advantages. However, low detection sensitivity and poor anti-interference capacity of these glucose-sensors seriously restrict their practical applications. In this regard, gas chromatography-mass spectrometry (GC-MS) [6] and liquid chromatography-mass spectrometry (LC-MS) [7], [8], [9], [10] approaches are introduced for blood glucose analysis because of their high accuracy and high sensitivity. However, it may be time-consuming because a gas- or liquid-phase separation process is required. The limitations of the current technology highlight the need for an accurate, sensitive and rapid method for the determination of blood glucose to produce convincing clinical measurements.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has become a crucial tool in the analysis of various macromolecules [11] due to its advantages of high throughput, high sensitivity, and low sample consumption [12]. However, very few MALDI MS methods [13], [14] have been reported for the analysis of glucose in blood because of background signal interferences in the small molecular region (< 700 Da) from organic matrices [15] Recently, our group proved black phosphorus as a new matrix for MALDI MS qualification and quantification of small molecule [16]. The research showed that black phosphorus assisted laser desorption/ionization-time of flight mass spectrometry (BP/ALDI-TOF MS) could produce a clean background during the analysis of small molecules. Moreover, the technique facilitated high detection sensitivity towards quaternary ammonium compounds. On the other hand, boronic acids can covalently bind with 1,2 or 1,3 cis-diols to form five- or six-membered cyclic esters [17], indicating that boronic acids can selectively bind to the cis-diols pairs of glucose. It inspired us to explore a reaction pathway for converting glucose to a quaternary ammonium compound via a chemical labeling reagent containing a boronic acid group and a quaternary ammonium moiety used in BP/ALDI-TOF MS detection. This may be a promising strategy for accurate, high–throughput, and sensitive measurements of glucose in clinical diagnosis applications.
Herein, a compound of 2-(4-boronobenzyl) isoquinolin-2-ium bromide (BBII) containing both boronic acid group and quanternary ammonium group was synthesized as the chemical labeling (CL) reagent. The CL-BP/ALDI-TOF MS strategy enabled a simple and high–throughput sample preparation procedure consisting a 1 min protein precipitation and a 3 min chemical labeling step before the MALDI MS analysis. In addition, due to the superior sensitivity of the detection method, glucose was successfully detected with a miniscule sample of 0.5 μL serum. With the proposed method, endogenous glucose in various serum samples were determined and quantified, and the results were in agreement with those obtained using the conventional clinical hexokinase method.
Section snippets
Chemicals and reagents
Glucose standard, ammonia hydrate (NH3·H2O, 25%, aqueous solution), formic acid (FA, 88%) were all obtained from Sinopharm Chemical Reagent (Shanghai, China). D-glucose-4,5,6,6′-d4 used as internal standard (IS), 4-(bromomethyl)phenylboronic acid and isoquinoline were all purchased from J&K Chemicals (Beijing). Acetonitrile (ACN, HPLC grade) was obtained from Tedia Co. (Fairfield, OH, U.S.A.). Purified water was obtained on the Milli-Q system (Milford, MA, USA). BP was purchased from XFNANO
General principle for the strategy
It was anticipated that high throughput, high sensitivity and low sample consumption exhibited by the MALDI MS would lend itself to the development of an accurate, rapid and sensitive method for detecting serum glucose. BP/ALDI-TOF MS has superior sensitivity for quaternary ammonium compounds, and the cis-diols pairs of glucose can bind well with a boronate reagent. Thus, we explored a reaction path way for converting glucose to a quanternary ammonium compound via a chemical labeling reagent
Conclusions
In summary, a reliable and simple CL-BP/ALDI-TOF MS strategy was developed for the rapid and sensitive analysis of glucose in blood serum samples. Endogenous glucose was selectively reacted with BBII and a CL-glucose derivative exhibited a strong response in BP/ALDI-TOF MS analysis. A simple protein precipitation procedure was needed before chemical labeling and MS detection, and the entire analysis of serum glucose was completed within 5 min. Moreover, due to the highly detection sensitivity of
Acknowledgments
The authors thank the National Natural Science Foundation of China (21475098, 21635006, 31670373), the Natural Science Foundation of Hubei Province, China (2014CFA002) and the Fundamental Research Funds for the Central Universities (2042016kf0035).
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