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Cite this: RSC Adv., 2015, 5, 41595
Synthesis, characterization and bifunctional applications of bidentate silver nanoparticle assisted single drop microextraction as a highly sensitive preconcentrating probe for protein analysis† Lokesh Shastri,a Hani Nasser Abdelhamid,b Mohd Nawaza and Hui-Fen Wu*acdef Synthesis, characterization and bifunctional applications of silver nanoparticles with two different surface capping reagents are reported. The surface engineering of AgNPs with 1-octadecanethiol (1-ODT)/4aminothiophenol
(4-AMP)
‘Ag@ODT/AMP’
and
1-octadecanethiol
(1-ODT)/1-thioglycerol
(1-TG)
‘Ag@ODT/TG’ were synthesized, characterized and applied for microextraction. The presence of two functional groups on the surface of AgNPs produced a multidendate that can interact with proteins and peptides such as insulin, heart cytochrome c, ubiquitin, lysozyme, cysteine, and homocysteine. Thus, they were applied in a single drop microextraction (SDME) process termed as silver nanoparticle assisted single drop microextraction (SASDME). The proteins after separation were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The effects of different parameters were optimized, such as sample pH, stirring rate, salt concentration, extraction time, matrix type, and the Received 9th March 2015 Accepted 22nd April 2015
amount of AgNPs. The present methodology has been successfully applied for the detection of insulin and cytochrome c in real samples (urine and milk) and for cysteine and homocysteine in a urine sample.
DOI: 10.1039/c5ra04032a
SASDME is a simple and effective microextraction technique for real sample analysis, which is rapid and
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possesses high sensitivity and selectivity.
Introduction Among different nanoparticles, silver nanoparticles (AgNPs) have been extensively studied for their synthesis, characterization and applications.1,2 Thus, they have been used for various applications in many elds. They have been used intensively for bioanalytical chemistry because of their unique properties, such as surface-plasmon resonance (SPR), larger surface area, catalytic properties, and quantum size effects, which contributes to the signal amplication of bioassays.3,4 It has been
a
Department of Chemistry, National Sun Yat-Sen University, Kaohsiung 70, Lien-Hai Road, Kaohsiung, 80424, Taiwan. E-mail:
[email protected]; Fax: +886-7525-3908; Tel: +886-7-5252000-3955
b
Department of Chemistry, Assuit University, Assuit 71515, Egypt
c
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan d
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University and Academia Sinica, Kaohsiung 80424, Taiwan
e
Center for Nanoscience and Nanotechnology, National Sun Yat-Sen University, 70, Lien-Hai Road, Kaohsiung 80424, Taiwan
f
Institute of Medical Science and Technology, National Sun Yat-Sen University 80424, Taiwan † Electronic supplementary 10.1039/c5ra04032a
information
(ESI)
This journal is © The Royal Society of Chemistry 2015
available.
See
DOI:
applied to a colorimetric assay for protein detection,5 separation,6 uorescence,7 electrochemical immunosensor,8 and antibacterial agents.1,2 Recent developments in the synthesis and functionalization methods for AgNPs are reviewed and highlighted in ref. 9. Sample-pretreatment methods such as separation or preconcentration prior to matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) analysis is highly desired.10–15 This would increase the sensitivity and improve the analysis of extremely low levels of different analytes in diverse matrices.10 Among the different preconcentration techniques, liquid–liquid extraction (LLE), in which the target analyte is concentrated using two immiscible liquid phases with or without NPs, is a highly used preconcentration method.11 In order to increase separation efficiency and selectivity, to reduce extraction time and for environmental concerns, nanoparticles were integrated for LLE. Thus, silver nanoparticles (AgNPs) have received tremendous attention as a separation surface for ionization and preconcentration for mass spectrometry.12–15 These techniques are very important for the analysis of protein biomarkers in order to get faster and effective disease diagnosis.10 Herein, we report the chemical engineering modication of AgNPs surface with two different capping agents using 1-octadecanethiol (1-ODT)/4-aminothiophenol (4-AMP) ‘Ag@ODT/
RSC Adv., 2015, 5, 41595–41603 | 41595
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AMP’ and 1-octadecanethiol (1-ODT)/1-thioglycerol (1-TG) ‘Ag@ODT/TG’. The nanomaterials were synthesized and characterized using TEM, SEM, FTIR, and UV-vis absorption. The materials were applied for the SDME of proteins such as bovine heart cytochrome c (MW 12.3 kDa, pI 9.5) and bovine pancreas insulin (MW 5.7 kDa, pI 5.3) in real samples. The data revealed that SASDME is simple, sensitive, selective and fast for protein separation in real samples such as urine and milk. The AgNPs have a bifunctional capability, and thus they can be used as effective concentrating probes for separation and also for surface assisted laser desorption/ionization mass spectrometry (SALDI-MS). The separation and identication of cysteine and homocysteine from a urine sample is also reported.
Experimental section Chemicals and materials All the chemicals used were of analytical reagent grade. Bovine heart cytochrome c (MW 12.3 kDa, pI 9.5), bovine pancreas insulin (MW 5.7 kDa, pI 5.3), a-cyano-hydroxycinnamic acid (CHCA) and triuoroacetic acid (TFA) were purchased from Sigma chemical Co. (St. Louis, MO, USA). Toluene and AgNO3 were obtained from Mallinchrodt chemicals (Phillipsburg, NJ, USA). 1-Thioglycerol (99%, GC) was obtained from Fluka chemicals (Steinheime, Germany). 1-Octadecanethiol (96%) and 4-aminothiophenol (97%) were purchased from Alfa Aesar (Johnson Matthey Company, Karlsruhe, Germany). For SDME, the microsyringe (1–10 mL) was purchased from Hamilton Company (Reno, NV, USA). All chemicals and reagents were prepared in deionized water that was puried using a Milli-Q reagent water system. Synthesis of the Ag nanoparticles modied with binary functional groups in toluene 0.9 mmol AgNO3 was dissolved in 200 mL water at 0 C. 0.45 mmol of a mixture of thiols was then added and stirred for 10 min. Typically, the ligands used were 1-octadecanethiol (1ODT) : 4-aminothiophenol (4-AMP)/1-thioglycerol (1-TG) in a 2 : 1 ratio in 20 mL of toluene, and then a saturated solution of NaBH4 was slowly added dropwise. Aer complete addition, the solution was stirred for 20 min then 2 mL HCl (1 M) was further added and stirred for 2 hours and placed in a refrigerator overnight to complete the reaction. The layer with dark brown color was collected using a separation funnel and washed with deionized water to remove the impurities. Instrumentation for characterization of AgNPs The UV-vis absorption spectra of bifunctionalized AgNPs were obtained on a double-beam UV-350 spectrophotometer at room temperature (Hitachi, Tokyo, Japan). The morphology and size of the AgNPs were evaluated using a scanning electron microscope (SEM, JEOL-6700) and a high resolution transmission electron microscope (HRTEM, JEOL TEM-3010, Tokyo, Japan). FTIR spectra were obtained using a Bruker IFS 66 v/s vacuum (Bruker, Germany) to conrm the surface modication. The spectra were collected over 500 scans at a resolution of 8 cm1.
41596 | RSC Adv., 2015, 5, 41595–41603
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MALDI-MS analysis All mass spectra were obtained in the positive ion mode using matrix-assisted laser desorption/ionization-time of ight-mass spectrometry (Microex, Bruker Daltonics, Bremen, Germany) with a nitrogen laser (337 nm). Ions were produced with a delayed extraction period of 200 ns and the accelerating voltage was set to 20 kV. All the experiments were carried out in a linear mode (>5000 Da) and reection mode (
