Single-drop microextraction for bioanalysis: present and future

Abstract

BioanalysisVol. 5, No. 21 EditorialFree AccessSingle-drop microextraction for bioanalysis: present and futureSuresh Kumar Kailasa & Hui-Fen WuSuresh Kumar KailasaDepartment of Chemistry, S. V. National Institute of Technology, Surat – 395007, Gujarat, India.Search for more papers by this author & Hui-Fen Wu* Author for correspondenceDepartment of Chemistry, & Center for Nanoscience & Nanotechnology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan and School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, 806, Taiwan and Doctoral Degree Program in Marine Biotechnology, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan. Search for more papers by this authorEmail the corresponding author at hwu@faculty.nsysu.edu.twPublished Online:1 Nov 2013https://doi.org/10.4155/bio.13.231AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: CEMALDI-MSnanomaterialssingle-drop microextractionIntensive efforts have been devoted to the development of miniaturized analytical tools for analysis of various biomolecules, such as proteins, mixtures of proteins and nucleic acids and protein digests. The separation and sensitive detection methods are the core of the field in proteomics, genomics and modern biology. The detection of biomolecules such as amino acids, peptides, proteins, nucleic acids and related substances in biological samples (blood, urine, tissues, cells and microorganisms) is collectively known as bioanalysis. Biological samples are complex mixtures, often containing salts, acids, bases and numerous organic compounds with similar chemical properties to those of the target analytes of interest [1]. Thus, sample preparation treatment is essential in extracting and enriching target biomolecules from the biocomplex matrices. With respect to this, various microextraction techniques related to liquid-phase microextraction and solid-phase microextraction have been integrated into bioanalytical methods/development for the extraction of biomolecules prior to their identification by MS. Miniaturization of extraction could simplify the sample extraction/preconcentration routes and time, and the acceptor phase can be easily integrated with the mass spectrometric instruments. Single-drop microextraction (SDME) is often used to represent the acceptor phase where only a small microdroplet (0.5–2.0 µl) is suspended on the tip of a microsyringe; while liquid–liquid microextraction (LLME) is used when the acceptor phase is >10 µl [2]. Among these liquid miniaturization tools, SDME has demonstrated superior ability in rapid extraction and preconcentration for target biomolecules with minimized volumes of sample solution (10–100 µl) and solvent (0.5–2.0 µl), and it can also be directly interfaced with MS such as GC–MS or MALDI-MS.The basic concept of SDME was first introduced by Cantwell et al. in 1996. They used organic drop (8.0 µl) as the acceptor phase, which is directly compatible with GC analysis [3]. After this invention, more efforts have been directed towards the development of various SDME approaches coupled with chromatographic and spectrometric techniques for the analysis of various types of analytes (inorganic, organic and biomolecules). Recently, several review papers have addressed the rapidly increasing rate of publications for SDME in a wide variety of biomolecule analysis [1–5]. Even the solvent consumption can be reduced to 99% when compared with traditional LLE. At present, SDME has been successfully combined with various analytical instruments (GC and HPLC), MS, atomic absorption spectrometry, CE and molecular spectroscopic techniques (UV–vis and fluorescence spectrometry) [4–6]. SDME can be classified into two modes: two-phase SDME (including direct-immersion SDME, continuous-flow SDME, drop-to-drop SDME and directly suspended droplet SDME); and three-phase SDME (such as headspace SDME and liquid–liquid–liquid SDME). The mathematical equations for the calculations of the equilibrium concentration of analytes in the organic phase and enrichment factor have been well-established [2,4,5]. “…larger surface-to-volume ratio of the acceptor phase leads to higher distribution coefficient of the analyte and to facilitate faster extraction with shorter time[2].”Green analytical chemistry is an aspect of green chemistry philosophy. It involves the development of miniaturized analytical techniques and analytical methodologies with minimized reagent and solvent consumption, and reduced extensive chromatographic separation for detection of biomolecules by various analytical instruments. In developing a green bioanalytical method, the sample pretreatment methods should be established with simplification for rapid extraction of target analytes from biological samples, by using minimal sample (reagent and solvent volumes) [2]. SDME has been practiced for many years with various designed methodologies with increasing degrees of analytical sophistication for biomolecule analyses by CE and MALDI-MS. A recent review on SDME summarized a compendium of developments in bioanalysis by CE [5]. However, CE requires a specific solvent system for the acceptor drop, since aqueous solvent or nonaqueous solvent may be utilized as a separation medium. Importantly, the extractant should be diluted to several microliters before CE injection. To solve this problem, the in-line SDME technique was fabricated and coupled with CE, while this approach was limited to only specific biomolecules analysis by CE. “MALDI-MS has been proved as the key technology for quantitative and qualitative analysis of biomolecules in proteomics and genomics”[7,8].Over the past 17 years, relentless efforts have been focused on SDME–MALDI-MS to extend its scope for a wide variety of chemical species extraction and preconcentration from various matrices. MALDI-MS has become a widespread bioanalytical technique due to its excellent features, such as rapidity, higher tolerance limit, large mass range, sensitivity and ease of operation. In SDME, the selection of solvent is crucial since too volatile solvents are not suitable. Typically, chemicals such as toluene, hexane, isooctane, octanol, n-octane, n-octyl acetate, isoamyl alcohol, undecane, octane, nonane and ethylene glycol are used as common solvents in SDME. The following features are necessary to indicate a suitable solvent in SDME coupled with MALDI-MS for bioanalysis: ▪ High boiling point with low vapor pressure to reduce evaporation;▪ Capable of dissolving the analytes to produce homogeneous crystals with the matrix;▪ Water-immiscible (organic solvent);▪ High viscosity to clench the microdrop, but not to affect the diffusion rate of analyte.These features are very important to ensure the excellent reproducibility and sensitivity of SDME combined with MALDI-MS. Importantly, the nature of analyte influences the SDME. For example, headspace-SDME is suitable for polar and nonpolar, low molecular weight, volatile and semivolatile compounds, while direct immersion SDME is more appropriate for the extraction of nonpolar or moderately polar higher molecular weight, semi-volatile chemicals. Sometimes, derivatization is essential for the highly polar molecules to ensure good recovery of target analytes. Furthermore, SDME efficiency is strongly dependent on the intermolecular attractions, such as London dispersion forces, van der Waals forces, electrostatic, hydrophobic, permanent dipole–dipole interactions, and hydrogen bonding. To date, 0.5–1.0 µl of drop volume has been typically used for SDME–MALD-MS; however, approximately 10 µl of drop volume was also used in some of the SDME modes coupled with other analytical instruments [2,4–5]. The microdrop stability was drastically decreased with the drop volume >3.0 µl. The drop volume is also reduced by the evaporation and/or dissolution in the sample matrix. Sample agitation plays a key role in enhancing SDME efficiency and minimizing the extraction time. So far, three types of sample agitation methods, namely stirring, vibration and vortexing, are used in SDME [2]. In SDME, the optimization of extraction time is very important in enabling the equilibrium of analytes between the aqueous solution with the organic drop. It was noticed that the signal intensities of analytes decrease with increasing time, which might be due to evaporation of solvent. However, the solvent properties (viscosity and volatile nature) are also influenced by the extraction time. In most of the SDME–MALDI-MS techniques, the magnetic stir bar was used and suitable stirring rates have been found at 120–360 rpm [2–5]. In the near future, special design of SDME methods will be proposed in order to extract large numbers of biomolecules from biological samples.In addition, sodium salts are often used with caution to enhance the extraction efficiency of SDME. But the addition of salts may not be beneficial for some of the organic nonpolar (semi-volatile) compounds. In contrast, salt additions can significantly enhance the extraction efficiency of SDME for biomolecules. We extensively studied the effect of salt addition in SDME for biomolecules (gramicidin D) and found that SDME efficiency greatly increased by adding 1.7 M of NaCl, which led to enhanced signal intensities of target biomolecules [9]. The SDME efficiency and signal intensities were increased due to the increase in ionic strength of solution and analytes having high affinity towards sodium ions, resulting in the generation of sodium adduct analyte ions with high intensities in the MALDI mass spectra. In the future, more efforts should focus on the automated SDME steps (agitation, syringe plunger movement, cleaning and injection) coupled with MALDI-MS for biomolecule analysis.In recent years, significant efforts have been made to integrate ionic liquids with SDME to enhance extraction efficiency. Ionic liquids exhibit unique physicochemical properties (negligible vapor pressure, good thermal stability, tunable viscosity and miscibility with water and organic solvents), which facilitate the use of larger drop volumes for longer extraction times than the conventional organic solvents [6]. The development of ionic liquids integrated with SDME coupled with MALDI-MS takes advantage of the knowledge and experience accumulated in inorganic ultratrace analysis; further studies in ionic liquid-assisted SDME for bioanalysis in the near future will be proposed. Logic organic framework on nanoparticles surfaces leads to magic analytical applications in SDME–MALDI-MS for the extraction, preconcentration and detection of biomolecules at ultra-trace levels.Clearly, integrative nanomaterials-based LLME–MALDI-MS is growing and leading to a better understanding of the quantity of biocomponents in biological samples at minimal volumes of samples and solvents. To our best knowledge, our group first introduced another intriguing technology on the horizon – termed ‘Nanoparticle-assisted SDME’, which has been demonstrated to be effective for biomolecules extraction and preconcentration prior to MALDI-MS [10]. The integration of metallic (Ag, Au and Pt) nanoparticles (NPs) in SDME allowed efficient extraction and preconcentration for biomolecules (peptides, proteins and bacterial proteins) at trace levels through electrostatic or hydrophobic interactions between NP surfaces and biomolecules, which led to signal enhancements in mass spectra [7, 10]. Integration of fabricated NPs in SDME has the following advantages in comparison with conventional SDME: ▪ Functionalized NPs act as probes for extracting and concentrating the target analytes at trace level from sample matrices;▪ Higher surfaces area of NP-dispersed organic droplets allow efficient preconcentration of target species through electrostatic or hydrophobic interactions;▪ Forming homogeneous crystals with target analytes, which facilitates the increase of the reproducibility and repeatability of MALDI-MS.Recently, our group demonstrated the utility and potentiality of Pt NP-dispersed ionic liquid as SDME solvent in extract, and in preconcentrating bacterial proteins in Escherichia coli and Serratia marcescens, which can then be detected by MALDI-MS at 107 cfu/ml [8]. These NP-assisted SDME combined with MALDI-MS approaches proved powerful techniques for extraction, preconcentration, separation and identification of various biomolecules (peptides, proteins and bacteria) with high sensitivity, excellent resolution and good reproducibility. Metal oxide (BaTiO3) and metal hydroxide (Mg[OH]2) NPs were also successfully integrated in LLME–MALDI-MS for bioanalysis [11,12]. The metal oxide NPs are ideal for integration in SDME–MALDI-MS for future bioanalysis. Several papers have described the potential applications of SDME coupled with various analytical instruments (UV-visible and fluorescence spectrometry, CE, GC, HPLC, GC–MS and LC–MS) for various chemical species; however, the merging of NP-based SDME coupled with MALDI-MS for bioanalysis is still in its infancy, and more approaches can be expected in the near future. In addition, some important issues such as automation, drop stability, multibicomponents extraction and preconcentration, and lack of hyphenation with MS bioanalysis remain to be addressed in the near future. Therefore, we will observe the continued and extremely rapid developments in this area, particularly with regard to miniaturized extraction techniques coupled with miniature MS to allow rapid extraction and preconcentration and to provide truly portable in situ MS bioanalysis capabilities. Improved miniaturized extraction tools combined with MS hypotheses can be derived by integrating different modes of needles, unique chemicals and nanomaterials.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1 Musteata FM. Extraction and microextraction in bioanalysis. Bioanalysis4(19),2321–2323 (2012).Link, CAS, Google Scholar2 Jeannot MA, Przyjazny A, Kokosa JM. Single drop microextraction – development, applications and future trends. J. Chromatogr. A1217,2326–2336 (2010).Crossref, Medline, CAS, Google Scholar3 Jeannot MA, Cantwell FF. Solvent microextraction into a single drop. Anal. Chem.68,2236–2240 (1996).Crossref, Medline, CAS, Google Scholar4 Alothman ZA, Dawod M, Kim J, Chung DS. Single-drop microextraction as a powerful pretreatment tool for capillary electrophoresis: a review. Anal. Chim. Acta739,14–24 (2012).Crossref, Medline, CAS, Google Scholar5 Choi K, Kim J, Chung DS. Single-drop microextraction in bioanalysis. Bioanalysis3(7),799–815 (2011).Link, CAS, Google Scholar6 Pena-Pereira F, Lavilla I, Bendicho C. Miniaturized preconcentration methods based on liquid–liquid extraction and their application in inorganic ultra-trace analysis and speciation: a review. Spectrochim. Acta Part B At. Spectrosc.64,1–15 (2009).Crossref, Google Scholar7 Shastri L, Kailasa SK, Wu HF. Nanoparticle-single drop microextraction as multifunctional and sensitive nanoprobes: Binary matrix approach for gold nanoparticles modified with (4-mercaptophenyliminomethyl)-2-methoxyphenol for peptide and protein analysis in MALDI-TOF MS. Talanta81,1176–1182 (2010).Crossref, Medline, CAS, Google Scholar8 Ahmadab F, Wu HF. Characterization of pathogenic bacteria using ionic liquid via single drop microextraction combined with MALDI-TOF MS. Analyst136,4020–4027 (2011).Crossref, Medline, Google Scholar9 Wu HF, Kailasa SK, Lin CH. Single drop microextraction coupled with matrix-assisted laser desorption/ionization mass spectrometry for rapid and direct analysis of hydrophobic peptides from biological samples in high salt solution. Rapid Commun. Mass Spectrom.25,307–315 (2011).Crossref, Medline, CAS, Google Scholar10 Sudhir PR, Wu HF, Zhou ZC. Identification of peptides using gold nanoparticle-assisted single-drop microextraction coupled with AP-MALDI mass spectrometry. Anal. Chem.77,7380–7385 (2005).Crossref, Medline, CAS, Google Scholar11 Kailasa SK, Wu HF. Dispersive liquid–liquid microextraction using functionalized Mg(OH)2 NPs with oleic acid as hydrophobic affinity probes for the analysis of hydrophobic proteins in bacteria by MALDI-MS. Analyst137,4490–4496 (2012).Crossref, Medline, CAS, Google Scholar12 Kailasa SK, Wu HF. Surface modified BaTiO3 nanoparticles as the matrix for phospholipids and as extracting probes for LLME of hydrophobic proteins in Escherichia coli by MALDI-MS. Talanta114,283–290 (2013).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByLiquid–Liquid Extraction of Organic Compounds into a Single Drop of the Extractant: Overview of Reviews29 July 2021 | Journal of Analytical Chemistry, Vol. 76, No. 8Applications of single-drop microextraction in analytical chemistry: A reviewTrends in Environmental Analytical Chemistry, Vol. 29Direct immersion single-drop microextraction of semi-volatile organic compounds in environmental samples: A reviewJournal of Hazardous Materials, Vol. 393Sample matrix influence on microdrop hold-up in single drop microextraction29 June 2020 | Acta Chimica Slovaca, Vol. 13, No. 1Practical sample pretreatment techniques coupled with capillary electrophoresis for real samples in complex matricesTrAC Trends in Analytical Chemistry, Vol. 122A green and efficient in-syringe ionic liquid-based single step microextraction procedure for preconcentration and determination of cadmium in water samplesJournal of Industrial and Engineering Chemistry, Vol. 27Nanomaterial-based miniaturized extraction and preconcentration techniques coupled to matrix-assisted laser desorption/ionization mass spectrometry for assaying biomoleculesTrAC Trends in Analytical Chemistry, Vol. 65 Vol. 5, No. 21 Follow us on social media for the latest updates Metrics History Published online 1 November 2013 Published in print November 2013 Information© Future Science LtdKeywordsCEMALDI-MSnanomaterialssingle-drop microextractionFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download