Immune-enrichment of insulin in bio-fluids on gold-nanoparticle decorated target plate and in situ detection by MALDI MS
© The Author(s) 2017
Received: 10 June 2016
Accepted: 6 January 2017
Published: 19 January 2017
Detection of low-abundance biomarkers using mass spectrometry (MS) is often hampered by non-target molecules in biological fluids. In addition, current procedures for sample preparation increase sample consumption and limit analysis throughput. Here, a simple strategy is proposed to construct an antibody-modified target plate for high-sensitivity MS detection of target markers such as insulin, in biological fluids.
The target plate was first modified with gold nanoparticle, and then functionalized with corresponding antibody through chemical conjugation. Clinical specimens were incubated onto these antibody-functionalized target plates, and then subjected to matrix assisted laser desorption ionization mass spectrometry analysis.
Insulin in samples was enriched specifically on this functional plate. The detection just required low-volume samples (lower than 5 µL) and simplified handling process (within 40 min). This method exhibited high sensitivity (limit of detection in standard samples, 0.8 nM) and good linear correlation of MS intensity with insulin concentration (R2 = 0.994). More importantly, insulin present in real biological fluids such as human serum and cell lysate could be detected directly by using this functional target plate without additional sample preparations.
Our method is easy to manipulate, cost-effective, and with a potential to be applied in the field of clinical biomarker detection.
KeywordsInsulin MALDI MS Antibody-enrichment Chemical conjugation
The level of insulin in the blood indicates explicitly the function of endocrine beta cells, and thus the metabolism situation of carbohydrates and fat. Thus, insulin is recognized as a marker protein for diagnosis of various types of diabetes and related diseases [1–6]. The detection of insulin in diabetes samples has been used in early-diagnosis, monitoring disease progression, prognosis and pathology research . The progression of technique for insulin analysis will undoubtedly improve the early diagnosis for relative disease and facilitate the follow-up therapy [3, 4].
Currently, insulin analysis is based on a number of methods, including chemiluminescence immunoassays , radio immunoassays , immune affinity chromatography-LC/MS/MS methods , surface plasma resonance immunosensors , capillary electrophoretic immunoassay , and immune enzymometric assays . However, most of these methods are time- and labor-consuming, of low analysis throughput, and use hazardous reagents such as the radioactive labels.
Matrix assisted laser desorption ionization mass spectrometry (MALDI MS) is a powerful tool for the detection and analysis of biomarkers . Owing to the remarkable features such as high-throughput, high sensitivity, label-free analysis and ease to manipulate, MALDI MS-based analysis has gained considerable interest and is now widely utilized in different fields of biomolecule analysis (including discovery, identification and monitoring), and even as a diagnostic platform [14–17]. Nevertheless, directly detecting low-level biomolecules such as insulin is hampered by the complex composition of real biological fluids. The concentrations of target peptide or protein in blood generally range from nM to μM , which were much lower than many other non-target molecules and thus very hard to detect or quantify. In addition, the performance of MS is usually hampered by the high content of salts in biological fluids, known as the effect of ionization suppression . Therefore, several immune-MALDI MS methods have been developed to pre-concentrate target analytes and increase analyzing efficiency. These methods generally applied immune-affinity column  or antibody-conjugated magnetic beads [21, 22] to capture target biomolecules, followed by MALDI MS analysis. However, these strategies required additional steps of centrifugation, sample transferring, or column-fractions, which increased sample consumption and limited the analysis throughput .
One solution is to specifically enrich target molecules on the MS target plate, to avoid additional handling processes such as column purification or sample transfer. A small number of studies have reported the use of this strategy to detect insulin [23–25]. In these reported methods, the antibody of target analyte was immobilized on 2-dimension planar substrate by using complicated chemical reagents [23, 24] or non-specific adsorption . However, the surface area of 2-dimension planar substrate was restricted, limiting the number of immobilized antibodies and the approach of target analytes [23–25]. Meanwhile, the complex organic chemicals such as dextran  may also introduce more non-target signals to MS analysis. Thus, the sensitivities of these strategies were generally in micromole range, hindering a more wide-spread application for the analysis of real biological fluids [23, 25]. Therefore, new design of target plate substrate is still needed to direct analyze real biological fluids using MADLI MS with minimum sample preparation.
Here, to achieve facilitated and sensitive insulin detection, we designed a simple strategy to immobilize insulin antibody directly onto a gold-nanostructured MALDI plate by means of chemical conjugation. The 3-dimension non-planar nano-surface increased surface area, and improved the efficiency of antibody binding, thus enhanced the sensitivity of analysis. Meanwhile, the simple small-molecular chemicals used in this platform avoided introducing interference signals in the MALDI MS. Compared with most previous immune-MALDI MS methods [20–22], this platform could simultaneously enrich and detect target insulin in biological fluids without any off-plate purification by affinity-beads/column, requiring a very small quantity of samples and oversimplified handling process.
3-Aminopropyltrimethoxysilane (APTMS), 2,5-dihydroxybenzoic acid (DHB), Trifluoroacetic acid (TFA), bovine serum albumin (BSA), human serum albumin (HSA), transferring, immunoglobulin G (IgG), Acetonitrile (ACN), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) hydrochloride were purchased from Sigma (St. Louis, Mo, USA). 11-mercaptoundecanoic acid (MUA) was obtained from Aldrich (Milwaukee, WI, USA). N-Hydroxysuccinimide (NHS) was purchased from Acros (New Jersey, USA). HAuCl4 was purchased from J&K Scientific Ltd. (Beijing, China). NaOH, NaCl, HCl, HNO3, absolute ethanol, methanol, ethanolamine (EOA) were of analytical reagent grade from Beijing Chemical Works (Beijing, China). Antibody of human insulin was purchased from the Cell Signaling Technology, Inc. ITO slides (10 Ω/sq) were purchased from Kaivo Electronic Components Co., Ltd. (Zhuhai, China). All reagents and solvents were used as received. Triply distilled water was used for the preparation of all solutions and rinsing.
Preparation of GNP
The gold nanoparticles (GNP) were prepared according to the previous literature . All glassware used was cleaned in aqua regia solution (HCl:HNO3 = 3:1) and then thoroughly rinsed by distilled H2O. Briefly, 100 mL of 0.01% HAuCl4 was heated to boiling in a round-bottom flask equipped with a condenser. Then 1.3 mL aqueous solution of sodium-citrate (1%) was added under vigorous stirring. In about 25 s, the solution turned blue; in approximately 1 min, the blue color change to red-violet gradually. After that the solution was kept boiling for an additional 10 min, and then cooled at room temperature. The prepared nanoparticle was stored at 4 °C.
Surface modification of ITO slide
Silylation of ITO slide
The ITO glasses were cut into 25 × 37.5 mm slides and cleaned by sequential sonication in acetone for 20 min, in isopropanol for 20 min, in soap water for 15 min, and twice in distilled water for 10 min. The cleaned ITO slides were immersed in 5 M NaOH for 8 h at room temperature and then flushed thoroughly with distilled water and dried under N2 stream. Afterwards they were immediately treated in a freshly prepared APTMS solution (3% in methanol) for 2 h. The resulting APTMS-ITO slides were sonically cleaned in methanol for three times and dried under N2 stream.
Deposition of GNP on APTMS-ITO
The APTMS-ITO slides were immersed in GNP solution for at least 8 h at 4 °C prior to flushing with distilled water and dried under N2 stream.
Derivatization of carboxyl group on GNP-ITO slide
The GNP deposited ITO (GNP-ITO) slides were immersed in a solution of MUA in ethanol (10 mM) for 8 h, rinsed with ethanol, and then dried under N2 stream.
Immobilization of protein on MUA-GNP-ITO
To covalently attach protein on these two kinds of substrate, an aqueous solution of EDC (75 mM) and NHS (15 mM) was first applied to treat the slides for 25 min at room temperature. Then the protein solutions (anti-insulin or BSA in pH 7.4 phosphate buffer) were spotted directly on the proper locations of the slides using pipettor. After all samples were spotted, the slides were laid in a sealed humid bottle at room temperature for at least 2 h to complete coupling reaction. Then an aqueous solution of EOA (1 M, adjusted with 5 M HCl to pH 8.6) was applied to treat the slides for 1.5 h to block the unreacted carboxyl groups. Finally, the anti-insulin or BSA modified ITO slides were flushed thoroughly with water to clean the unbound proteins and dried under N2 stream.
Insulin immune-reaction and MALDI-TOF analysis
Human insulin (0–72 nM) was dissolved in pH 7.4 phosphate buffer (10 mM) or PBS buffer containing albumin (35 mg/mL), transferring (2 mg/mL) and IgG (6 mg/mL) to generate standard insulin solutions. Five microliter of insulin solution was dropped on the anti-insulin modified ITO slide and incubated on a shaking table at room temperature for 30 min. Then the ITO plate was rinsed with Tween 20 solution (0.05% Tween 20 in water) and distilled water sequentially and dried under N2 stream. After that, 1 μL of DHB (15 mg/mL, 50% ACN, 0.1% TFA) was applied as the matrix for MALDI-TOF mass analysis. The solvent was dried naturally under room temperature and then the target plate was subjected to MALDI MS analysis.
Mass spectrometry was acquired on SHIMADZU AXIMA Resonance MALDI-IT-TOF on reflective/positive ion mode. Laser power of 105 mV was selected as standard desorption energy in MS analysis.
Results and discussion
Strategy to anchor insulin antibody on MALDI MS target plate
An indium tin oxide coated glass slide (ITO slide) was selected as MALDI MS target plate due to its ease of chemical modification and excellent conductivity , the latter is important for the efficient MALDI MS analysis . An easy approach to construct nano-surface was designed, and used to immobilize the insulin antibody on the ITO slide by chemical conjugation.
Sensitivity of the MALDI MS based on the anti-insulin modified GNP-ITO
Repeatability of MALDI MS results obtained on antibody-modified GNP-ITO in 6 patient serum samples (accepting insulin therapy)
Mear (MS intensity)
CV% for within day
CV% for between day
Analysis of biological fluids
Insulin levels in 6 human serums tested by chemiluminescence immunoassays (CIA) and the developed immune-MS method
Serums of patients (accepting insulin therapy)
Here, we proposed a strategy to construct antibody-functionalized MALDI target plate for fast and high-throughput MS analysis of insulin. The nanostructure on the surface of target plate improved the efficiency of antibody coupling and insulin detection. Biological fluids, such as serum or cell lysate, could be sensitively analyzed using this functional target plate without any pre-purification, which is impossible for the conventional MALDI MS methods. Furthermore, our method is also useful for the immobilization of other antibodies and analysis of other types of biomolecules, thus offering a widely-used toolbox for the fields of bio-sample exploration.
albumin bovine serum
indium tin oxide
- MALDI MS:
matrix assisted laser desorption ionization mass spectrometry
KL carried out the construction of functionalized target plates, MALDI MS experiments, data analyses and participated in drafting the manuscript. HW contributed to the preparation of standard, clinical and animal samples, and clinical information. YL conceived the study, contributed to its design and coordination, participated in drafting the manuscript and critical review. All authors read and approved the final manuscript.
Help from Torsten Juelich in Peking University for assistance with discussion and editing is greatly acknowledged.
The authors declare that they have no competing interests.
Availability of data and materials
The authors declare that all the data and materials are available.
Ethics approval and consent to participate/publication
A total of 6 serum samples were collected at the time of diagnosis from Peking University First Hospital after approval by the Ethic Review Board of the hospital. All patients gave written informed consent for study participation and consent for publications to this hospital (IRB PA20140929).
This work was supported from the Major State Basic Research Development Program of China (973 Program), Grant 2013CB910100 (Y. L.), and the Key Research Program of the Chinese Academy of Sciences, Grant KJZD-EW-TZ-L05 (Y. L.).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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