Abstract
Immunoassays anvendes til at opdage proteiner baseret på tilstedeværelsen af associerede antistoffer. På grund af deres omfattende brug i forskning og kliniske indstillinger, kan findes et stort infrastruktur af immunoassay instrumenter og materialer. For eksempel, 96- og 384-godt polystyrenplader er tilgængelige kommercielt og har et standard design til at rumme ultraviolet-synligt (UV-Vis) spektroskopi maskiner fra forskellige producenter. Desuden er en bred vifte af immunoglobuliner, påvisningstags og blokerende midler for kundespecifikke immunoassay designs som enzymbundne immunosorbent assays (ELISA) er tilgængelige.
På trods af den eksisterende infrastruktur, behøver standard ELISA kits ikke opfylder alle forskningsbehov, der kræver individuel immunoassay udvikling, hvilket kan være dyrt og tidskrævende. For eksempel ELISA kits har lav multiplexing (detektion af mere end én analyt ad gangen) kapaciteter da de afhænger normalt fluorescens eller colorimetric metoder til påvisning. Kolorimetrisk og fluorescerende-baserede analyser har begrænset multiplexing kapaciteter grundet brede spektrale toppe. I modsætning hertil Ramanspektroskopi-baserede metoder har meget større kapacitet til multiplexing grund smalle emissionstoppe. En anden fordel ved Raman spektroskopi er, at Raman reportere oplever signifikant mindre fotoblegning end fluorescerende tags 1. På trods af de fordele, som Raman reportere har over fluorescerende og kolorimetriske mærker, at protokoller fabrikere Raman-baserede immunoassays er begrænsede. Formålet med dette dokument er at give en protokol til at forberede funktionaliserede sonder til brug i forbindelse med polystyren plader til direkte påvisning af analytter ved UV-Vis analyse og Raman spektroskopi. Denne protokol vil give forskere til at tage en gør-det-selv tilgang til fremtidig multi-analyt detektion mens udnytte forud fastsat infrastruktur.
Materials
| Name | Company | Catalog Number | Comments |
| 60 nm Gold Nanoparticle | Ted Pella, Inc. | 15708-6 | These are citrate capped gold nanoparticles. Please see Discussion for relationship between Raman reporter and AuNP surface charge and its imporance to proper selection of AuNP and/or Raman reporter. |
| Sodium Bicarbonate | Fisher Scientific | S233-500 | |
| Methanol | Pharmco-Aaper | 339000000 | |
| Tris Buffered Saline (10x) pH 7.5 | Scy Tek | TBD999 | |
| Bottle Top Filtration Unit | VWR | 97066-202 | |
| Tween 20 (polysorbate 20) | Scy Tek | TWN500 | Used as an emulsifying agent for washing steps. |
| Phosphate Buffered Saline 10x Concentrate, pH 7.4 | Scy Tek | PBD999 | |
| Protein LoBind Tube 2.0 ml | Eppendorf Tubes | 22431102 | LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes. |
| Protein LoBind Tube 0.5 ml | Eppendorf Tubes | 22431064 | LoBind tubes prevent binding of proteins and AuNPs to surfaces of the tubes. |
| Microplate Devices UniSeal | GE Healthcare | 7704-0001 | Used for sealing and storing functionalized plates. |
| Assay Plate, With Low Evaporation Lid, 96 Well Flat Bottom | Costar | 3370 | |
| HPLC grade water | Sigma Aldrich | 270733-4L | |
| 3,3′-Diethylthiatricarbocyanine iodide (DTTC) | Sigma Aldrich | 381306-250MG | Raman reporter |
| mPEG-Thiol, MW 5,000 - 1 gram | Laysan Bio, Inc. | MPEG-SH-5000-1g | |
| OPSS-PEG-SVA, MW 5,000 - 1 gram | Laysan Bio, Inc. | OPSS-PEG-SVA-5000-1g | OPSS-PEG-SVA has an NHS end. |
| Mouse IgG, Whole Molecule Control | Thermo Fisher Scientific | 31903 | Antigen |
| Goat anti-Mouse IgG (H+L) Cross Adsorbed Secondary Antibody | Thermo Fisher Scientific | 31164 | Antibody |
| Human Serum Albumin Blocking Solution | Sigma Aldrich | A1887-1G | Bovine serum albumin can be used instead. |
| Mini Centrifuge | Fisher Schientific | 12-006-900 | |
| UV-Vis Spectrophotometer | Thermo Scientific | Nanodrop 2000c | |
| UV-Vis Spectrophotometer | BioTek | Synergy 2 | |
| Desalting Columns | Thermor Scientific | 87766 | |
| In-house built 785 nm inverted Raman microscope unit | N/A | N/A | An inverted Raman microscope is best for proper focusing onto surface of the well plate. Otherwise a very low magnification will be used due to height of the 96-well plate. An in-house built system was used as it was cheaper than buying from a vendor. However, any commercially available inverted Raman microscope system can be used. |
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