Conditionally activating optical contrast agent with enhanced sensitivity via gold nanoparticle plasmon energy transfer: feasibility study

Abstract

Background: Molecular sensing/imaging utilizing fluorophores has been one of the most frequently used techniques in biomedical research. As for any molecular imaging techniques, fluorescence mediated sensing always seeks for greater specificity and sensitivity. Since fluorophores emit fluorescence while their electron energy state changes, manipulating the local electromagnetic field around the fluorophores may be a way to enhance the specificity and sensitivity. Gold nanoparticles (GNPs) are known to form a very strong electromagnetic field on their surface [i.e., surface plasmon field (SPF)], upon receiving photonic energy. The level of fluorescence change by GNP-SPF may range from complete quenching to extensive enhancement, depending upon the SPF strength, excitation and emission wavelengths, and quantum yield of the fluorophore.

Method: Here, we report a novel design that utilizes BOTH fluorescence quenching and enhancement abilities of the GNP in one single nano-entity, providing high specificity and sensitivity. The construct utilizes a specially designed molecular dual-spacer that places the fluorphore at the location with an appropriate GNP-SFP strength before and after exposed to the biomarker. A model system to test the concept was an optical signal mediator activated by urokinase-type plasminogen activator (uPA; breast cancer secreting enzyme).

Results: The resulting contrast agent shows less than 10% of the natural fluorescence but, in the presence of uPA, its fluorescence emission is triggered and emits its fluorescence approximately twice of the natural form.

Conclusion: This study demonstrated that our novel design of an optical contrast agent can be conditionally activated with enhanced sensitivity, using both quenching and enhancement phenomena of fluorophores in the electromagnetic field of the appropriate strengths (in this case, locally generated by the GNP-SPF). This entity is similar to molecular beacon in terms of specificity but with greater sensitivity. In addition, it is not restricted to only DNA or RNA sensing but for any designs that cause the change in the distance between the fluorophore and GNP, upon the time of encountering biomarker of interest.

Figure 1

A schematic diagram illustrating the fluorescence emission level with change in the distance between a fluorophore and a GNP. Outside the SPF field of a GNP, fluorescence level does not get affected. As the fluorophore gets closer to the GNP, fluorescence is enhanced until it reaches a particular distance (LL). If the distance becomes even closer the fluorescence gets quenched, and on the GNP surface (SS) the fluorescence becomes completely quenched.

Figure 2

Enzyme-biomarker triggered, highly sensitive fluorophore/GNP complex. The complex normally emits little fluorescence because the SS places the fluorophore close to the GNP for fluorescence quenching. When the complex is placed in an environment with the enzyme biomarker, SS is cleaved by the enzyme and the distance between the fluorophore and GNP becomes to the length of LL, resulting in the fluorescence emission at an enhanced level. In this illustration, to simplify the concept, only a set of SS and LL for the GNP are shown. In reality, multiple SS/LL-fluorophore sets are to bind to a single GNP.

Figure 3

Structures of ICG, Cypate, and modified Cypate (mCy).

Figure 4

Theoretically estimated fluorescence level of Cypate (Ex/Em, 780/830 nm) relative the fluorescence without the influence of GNP-SPF, with change in the distance from a GNP at sizes 5, 10, 15, and 30 nm.

Figure 5

Confirmation of SS integrity. (A) Relative fluorescence levels of the GNP-SS-Cypate for the GNP size of 3.7, 8.0, and 16.4 nm. With 3.7 and 8 nm GNPs, the fluorescence is quenched significantly. For 16.4 nm, the fluorescence is enhanced, instead. (B) Fluorescence of 8.0 nm GNP-SS-Cypate before and 5 minutes after adding uPA. Fluorescence is restored as uPA cleaves SS.

Figure 6

Relative fluorescence of GNP-LL-Cy conjugated to 8 nm GNP, for the LL spacer that provids good flourescence enhancement for our purpose. Fluorescence is enhanced approximately twice of the level by Cypate alone.

Figure 7

An issue in designing two-spacer NanoPPET. (A) Idealistic two spacer conjugation for Cypate-dual spacer–GNP complex and (B) the product likely formed during the Cypate and GNP reacting with two spacers: GNP cross-linking and precipitation.

Figure 8

Chemical structure of the new dual spacer. The modification was reducing the number of binding sites from four sites (two between the spacers and Cypate, and two between spacers and GNP) to two (one to Cypate and one to GNP), by converting the design to a ring-shaped dual spacer (rSP).

Figure 9

Characterization of resulting uPA-specific NanoPPET. (A) Absorption spectra of 8.0 nm GNP, GNP-SCOAT, GNP-rSP-mCy, and mCy and (B) Fluorescence emission levels for free mCy, GNP-rSP-mCy complex, and uPA treated complex.