Fremstilling af en UV-Vis og Raman spektroskopi Immunoassay Platform

Summary

Nanoparticle-based optical probes have been designed as a vehicle for detecting antigens using Raman and UV-Vis spectroscopy. Here we describe a protocol for preparing such probes for a UV-Vis/Raman spectroscopy immunoassay in such a way to incorporate future multiplexing capabilities.

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.

Introduction

Typical sandwich immunoassays indirectly detect the presence of an antigen using two antibodies. The capture antibody is bound to a solid surface and forms an antibody-antigen complex when in proximity to an appropriate antigen. A detection antibody is then introduced and binds to the antigen. After washing, the antibody/antigen/antibody complex remains and is detected by the labeled detection antibody as demonstrated in Figure 1A. Typical detection is done by a fluorescent or colorimetric detector, limiting multiplexing to 10 analytes due to broad spectral peaks^2,3. In contrast, Raman-based systems have much narrower emission peaks resulting in enhanced multiplexing capabilities with sources claiming simultaneous detection of up to 100 analytes^2,3.

Many literature sources are available which cover important aspects related to immunoassays^4–6 such as step-by-step details to create personalized ELISA kits. Unfortunately, these protocols are for fluorescent or colorimetric detection, limiting multiplexing capability of customized immunoassays. To address this need, we present a detailed procedure to fabricate the UV-Vis/Raman immunoassay published previously⁷ for a direct immunoassay as illustrated in Figure 1B.

This protocol includes the fabrication of functionalized gold nanoparticle-based probes, illustrated in Figure 2. The procedure to make the Raman/UV-Vis probes begins by binding Raman reporters to the surface of gold nanoparticles (AuNPs). The AuNPs are then functionalized with antibodies that are associated with polyethylene glycol (PEG). Remaining binding sites on the AuNPs are blocked by binding methoxy polyethylene glycol thiol (mPEG-SH) to AuNPs to prevent subsequent non-specific binding during analysis. The prepared AuNP probes are tested by binding to antigens fixed to the wells of a polystyrene plate as illustrated in Figure 1B. Upon washing the plate, the AuNP probes are detected using UV-Vis spectroscopy while the associated Raman reporters are detected with Raman spectroscopy. Combining UV-Vis and Raman spectral data provides two methods of analyses, enhancing the capabilities of this immunoassay.

Protocol

1. Preparation of Buffers Phosphate Buffered Saline (PBS) Dilute 50 ml of 10x PBS with 450 ml HPLC grade water to make a 1x PBS concentration. Sterile filter the solution with a 0.22 µm filter. Store solution at room temperature. Preparation of Tris Buffered Saline + Tween 20 (TBST) Dilute 50 ml of 10x Tris Buffered Saline (TBS) with 450 ml HPLC grade water to make a 1x concentration. Add 250 μl of Tween-20 …

Representative Results

In this study, 60 nm gold particles were used for UV-Vis spectroscopy. UV-Vis absorption spectra from 400 to 700 nm were collected and the peak areas for each AuNP concentration were determined using an open source spectral analysis software8. Prior to peak integration, the collected spectra underwent baseline correction using a three-point polynomial fit. Peak areas were used to generate a logarithmic calibration curve as demonstrated in Figure 4. It should be noted that Figures 4 </s…

Discussion

In the detailed protocol, there are several critical points to address. One issue is the choice of Raman reporter and gold nanoparticle. Although the protocol was written to be adapted for individual use, the Raman reporter DTTC was used as an example. DTTC is a positively charged reporter and binds to negatively charged surfaces such as citrate capped AuNPs. This protocol can be adapted for negatively charged reporters by using gold nanoparticles with a positive surface charge. For example, polyethyleneimine (PEI) cappe…

Materials

60nm 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.
In-house built 785nm 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.
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