دانلود مقاله ترجمه شده افزایش انتقال انرژی رزونانس فورستر ریز بلور های کوانتوم در ماتریس های کاغذی – مجله IEEE

فهرست مطالب:

چکیده
مقدمه
۲ ریز بلور های کوانتوم به عنوان پروب های FRET محور
۳ مواد و روش ها
رسوب گام به گام QDs و پپتید ها
رسوب کانژوگیت های پپتیدی QD مونتاژ شده
اندازه گیری های PL
آنالیز های پروتولیتیک
۴نتایج و بحث
روش های رسوب گذاری
جفت QD-A555 FRET
FRET روی سوبسترای کاغذی
میکروسکوپ Fluorescence Lifetime Imaging
تست های پروتولیتیک
نتیجه گیری

بخشی از ترجمه:

کریستال های کوانتوم نیمه های شفاف نقش فزاینده و قابل توجهی در تحقیقات بیوفوتونیک و زمینه های کار بردی از جمله رد یابی های زیستی ایفا می کنند. در این جا ما روش های رسوب QDs را روی فیبر های سلولز با سوبسترای کاغذی جهت ردیابی FRET محور انتقال انرژی رزونانس فورستر ارایه می دهیم. تعیین خصوصیات حالت پایدار و فلورسانس زمانی FRET بین QDs رسوب یافته و پپتید های رنگی بر چسب گذاری شده درون ماتریکس کاغذی پیشرفت و بهبود قابل توجهی را در کارایی انتقال انرژی نشان داد. در مقایسه به محلول کل اولیه سرعت انتقال انرژی تقریبا ۴۰ برابر افزایش پیدا کرد و موجب افزایش همزمان ۷ برابری در نسبت تصاعد رنگی گیرنده حساس FRET شده و در نهایت از تصاعد QD جلوگیری به عمل آورد. لکه های QD رسوب یافته با مقدار مختلف پپتید های رنگی دارای روشنایی خفیفی تحت اشعه دهی فرابنفش بود و این که QD خالص و تصاعد A555 توسط چشم به صورت رنگ های مختلف قابل رویت بود. جذب و هضم تریپتیک پپتید های لینک کننده رنگ گیرنده و دهنده موجب کاهشFRET. شد.تغییر در نسبت رنگی به QD PL موجب رد یابی فعالیت پروتولتیک شامل اثرات افزایشی مقدار اپروتینین به عنوان باز دارنده بالقوه تریپسین بود. ترکیب QD – سوبسترای کاغذی و FRET دارای پتانسیل زیادی برای توسعه رد یابی های زیستی می باشد.

۱ مقدمه

خواص الکترونیک و اپتیکی منحصر به فرد نانوذرات مختلف(NPs) همواره مورد توجه بسیاری از تحقیقات بیوفوتونیک در سال های گدشته بوده است. مواد متنوعی نظیر آلو تروپ های کربن، (NPs) های فلزات جدید و نانو خوشه ها، (NPs) های لانتانید و ریز بلور های کوانتوم نیمه رسانا به عنوان پروب های اپتیکی برای تصویر برداری سلولی، ترانوستیک و تشخیص و رد یابی های سلولی مورد استفاده قرار گرفته اند. در هر یک از موارد خواصی نظیر اندازه، مساحت ویژه سطحی، ظرفیت حمل، و جذب و تصاعد نور دارای مزیت های کارکردی قابل توجهی بوده اند. برای رد یابی های آزمایشگاهی و تشخیص های آن، (NPs) می توانند حساسیت فوق العاده، استحکام و فعالیت زیادی داشته باشند به خصوص اگر با پروب های مختلف بیومولکولی و ظرفیت بالای تکثیر تیمار شوند.

بخشی از مقاله انگلیسی:

Abstract—Brightly luminescent semiconductor quantum dots (QDs) continue to have an increasing role in biophotonic research and applications such as bioassays. Here, we present methods for the immobilization of QDs on the cellulose fibers of paper substrates for Förster resonance energy transfer (FRET)-based assays of proteolytic activity. Steady-state and time-resolved fluorescence characterization of FRET between immobilized QDs and self-assembled dye-labeled peptides within the paper matrix revealed a substantial enhancement in energy transfer efficiency. Compared to bulk solution, the rate of energy transfer increased approximately 4-fold resulting in a concomitant 7-fold increase in the ratio of FRET-sensitized acceptor dye emission and quenched QD emission. Spots of immobilized QDs with different amounts of dye-labeled peptide had bright luminescence under UV/violet illumination and the net QD and A555 emission was visible by eye as different colors. Tryptic digestion of the peptide linking the QD donor and acceptor dye resulted in loss of FRET. Changes in the dye/QD PL ratio permitted tracking of proteolytic activity, including the effect of increasing amounts of aprotinin, a potent inhibitor of trypsin. The combination of QDs, a paper substrate, and enhanced FRET has strong potential for developing bioassays. Index Terms—Quantum dots, fluorescence, Förster resonance energy transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), paper diagnostics, biosensors, proteases, inhibition assay. I. INTRODUCTION HE unique optical and electronic properties of various nanoparticles (NPs) have been a major focus of biophotonic research in recent years. Materials as diverse as carbon allotropes [1, 2], noble metal NPs and nanoclusters [3, 4], lanthanide NPs [5], and semiconductor quantum dots (QDs) [6], among many others, have been used as optical probes for cellular imaging, theranostics, and in vitro assays and diagnostics. In each case, properties such as size, surface area, cargo capacity, and light absorption and emission have provided distinct functional advantages. For in vitro assays The authors thank the Natural Sciences and Engineering Research Council of Canada (NSERC), the Peter Wall Institute for Advanced Studies (PWIAS), the Canada Foundation for Innovation (CFI), and the University of British Columbia for support of this research. H. Kim was supported by an NSERC undergraduate research award. E. Petryayeva was supported by an NSERC postgraduate fellowship. W.R. Algar is supported by a Canada Research Chair (Tier 2). We thank Cheryl Ng for assistance with some experiments. All authors are with the Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada (algar@chem.ubc.ca). and diagnostics in particular, NPs can offer enhanced sensitivity [7-9], robustness, higher avidity and activity when functionalized with multiple biomolecular probes [10, 11], and greater capacity for multiplexing [12]. Although advances in the capabilities and availability of both NP materials and sophisticated optical technologies have led to the development of better bioassays, the accessibility and practicality of many of these approaches can be limited to advanced research and clinical laboratories. There is a significant need for simple, low-cost, portable, and robust bioassays that are suitable for lower resource settings such as field deployment, patient point-of-care, and use in developing countries. To address these needs, there has been a surge in research toward test strips, lateral flow assays, and micropaper analytical devices (µPADs) [13-15]. Many recent efforts have sought to incorporate NPs and their associated advantages into these technologies. In particular, gold NPs have been extensively utilized [16], largely due to their facile synthesis, intense coloration, red-to-blue color change upon aggregation [9], and silver enhancement methods [17]. In comparison, low-cost assays with QDs have been much less investigated, albeit that a few lateral flow immunoassays [18- 20] and paper-based assays [21, 22] have been reported. Here, we also investigate the potential for paper-based assays with QDs through both fundamental spectroscopic characterization and an example of a bioassay for proteolytic activity and inhibition. While not without potential challenges, paperbased assays with QDs are a promising avenue for research given the widely reported capabilities of QDs in other bioimaging and bioanalysis formats. II. QUANTUM DOTS AS FRET-BASED BIOPROBES QDs have emerged as important labels for cellular, tissue, and single molecule imaging [12, 23, 24]. The principal advantages of QDs in these applications are their broad absorption spectra, large one-photon (104 –۱۰۷ M–۱ cm–۱ ) and two-photon (103 –۱۰۴ GM) absorption coefficients, narrow and symmetric PL emission spectra (25–۳۵ nm full-width-at-halfmaximum, FWHM), the ability to tune the PL emission wavelength through nanocrystal size and composition, and resistance to photobleaching [6, 25]. These properties also offer superior performance and more straightforward implementation of multicolor imaging. QDs can be modified with antibodies or other affinity ligands to target various biomarkers, cells, and tissues. Another prominent area of interest that benefits from the above properties of QDs is the development of Förster resonance energy transfer (FRET)-based probes where changes in PL intensity are used to signal the presence or activity of biological targets [26, 27]. The most common role of QDs in FRET systems is as donors. The narrow PL of the QD allows the spectral overlap with an acceptor chromophore to be maximized while also minimizing crosstalk when measuring both donor and acceptor emission. Moreover, the ability to excite QDs across a broad range of wavelengths allows direct excitation of acceptors to be minimized. The FRET efficiency can also be systematically enhanced by arraying multiple acceptors per QD. Various dark quenchers [28, 29], fluorescent dyes [29, 30], fluorescent proteins [7, 31, 32], and gold nanoparticles [33, 34] have been paired as acceptors with QD donors. Recent studies have further demonstrated energy transfer between QDs and carbon nanomaterials [35-37]. To a much lesser but nonetheless growing extent, QDs have also been utilized as acceptors for luminescent lanthanide complexes [38, 39], chemiluminescent species [40], and bioluminescent proteins [41]. QD-FRET probes have been developed for a multitude of different biological targets and operate through a variety of transduction mechanisms [26, 27]. One such mechanism is associative, where a biorecognition process generates the proximity needed for FRET. Examples have included sandwich immunoassays [42] and sandwich hybridization assays [8]. In contrast, a dissociative mechanism relies on a biorecognition process eliminating the proximity required for FRET, as demonstrated with hydrolytic enzymes such as proteases [29] and nucleases [43]. Competitive binding assays have been demonstrated with QDs and FRET for nutrients [44], DNA [45], and RNA [46], among other targets. Unlabeled endogenous target typically displaces dye-labeled exogenous target in these assays. As another transduction mechanism, changes in biomolecular conformation upon biorecognition are often utilized to modulate donor-acceptor distance and FRET efficiencies without complete dissociation. This format is epitomized by QD-based molecular beacons for DNA detection [47]. More recently, QD-FRET probes for detecting changes in biological environments have been developed on the basis of changes in spectral overlap between QD donors and dye acceptors, as demonstrated with pH sensing [48]. Detection of some analytes is also possible by altering acceptor re-emission rather than FRET efficiency. This type of format has been demonstrated for reagentless maltose sensing [49] and oxygen sensing [50]. In addition to the diverse targets already mentioned, energy transfer-based QD probes have recently been reported for glucose and galactose [51], cocaine [52], kinase and phosphatase activity [53, 54], fluoride [55], mercuric ion [56], renin [57], carcinoembryonic antigen [58], mucin 1 [59], and many other analytes too numerous to mention here. The vast majority of the above assays have had a homogeneous format (i.e. solution phase). Comparatively few heterogeneous (i.e. solid phase) FRET-based assays have been developed with QDs. The few examples have included protease assays on glass chips [34] and nucleic acid hybridization assays on optical fibers [60], within microtiter plate wells [61], or, most recently, on paper substrates [21]. In this study, we present and evaluate three different methods for the immobilization of QDs within a paper matrix, compare FRET between solution and paper environments, and demonstrate a paper-based protease inhibition assay. Alexa Fluor 555 (A555) labeled peptides are self-assembled to CdSeS/ZnS core/shell QDs in bulk solution (Fig. 1A) and within the paper matrix (Fig. 1B). For the latter, QDs are immobilized through self-assembly after chemically modifying the cellulose fibers of the paper with ligands (Fig. 1C) that have high affinity for the ZnS shell of the QDs. Within the paper matrix, substantial increases in the relative Copyright (c) 2013 IEEE. Personal use is permitted. For any other purposes, permission must be obtained from the IEEE by emailing pubs-permissions@ieee.org. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. JSTQE-INV-NB-04976-2013 3 amount of FRET-sensitized A555 emission and the FRET efficiency are observed using steady-state and fluorescence lifetime measurements. When the A555-labeled peptide sequences are designed to be a substrate for a protease such as trypsin (TRP), it is possible to detect proteolytic activity through changes in FRET and measure the effect of an increasing amount of a protease inhibitor such as aprotinin. Our results show that the combination of QDs, a paper matrix, and the concomitant enhancement in FRET is suitable for developing sensitive assays for proteolytic activity.