Label-Free and Labeled Technology for Protein Characterization and Quantitation

September 1, 2010

Volume 23, Issue 9

Page Number: 40–45

Use it label-free, or add labels to detect contaminants in solution.

ABSTRACT

This article presents a label-free platform for protein characterization and quantitation based on localized surface plasmon resonance (LSPR) on a nanostructured metallic film. With recent advances in manufacturing techniques, the reproducibility of nanostructured thin films has allowed the transition of LSPR from academic interest into the first system commercialization for practical use in research, bioprocess, and diagnostic applications. The nanostructured gold films exhibit a distinct color caused by the absorption of certain wavelengths in the spectrum of white light. The film color changes as biomolecules or other chemicals come into contact with the LSPR surface, enabling precise quantitation of biomolecular interactions. This article reviews the sensitivity and dynamic range of LSPR. It also explains how LSPR signals can be amplified through enzymatic reactions to achieve greater and faster sensitivities than published results for ELISA end-point analyses.

Stained glass has been displayed in the windows of churches all across Western Europe since Medieval times. It was only in 1857 that Faraday1, and later Rayleigh and Mie,2 formulated a scientific explanation for the coloration of the stained glass. As it turns out, the stained glass colors are based on the sands used by the ancient artisans. Unknown to them, different sands contained various quantities of metal salts or minerals (such as gold chloride, gold oxides, cobalt, and silver compounds) which, when melted into glass, resulted in distinct colors ranging from reds and blues to yellow. We now understand that the metal minerals decomposed in the melt and the metal ions aggregated into nanometer-size inclusions. These glass colors are based on wavelength absorbtion of light by the electronic oscillation modes of the metal nanoparticles, called localized surface plasmons.3

LamdaGen Corporation

Basic studies using colloidal solutions showed that colors and wavelengths absorbed can be tailored by the metal's nature, size, and local environment surrounding the metal colloids.4 Using colloidal solutions to monitor binding events for label-free bioanalyses has become routine.5 However, the transition from the academic laboratory to commercialization has been awaiting manufacturing techniques that can replicate what researchers have achieved in Eppendorf tubes.6 With the emergence of powerful metrology tools that can explore the nanoworld, the relationship between the nano and the macro realm finally could be mastered. Today, stable metal films with precise nanostructuring and tunable absorption can be routinely manufactured using a wide range of surfaces. These films are at the core of a new commercial technology, termed localized surface plasmon resonance (LSPR). This article aims to introduce the salient features of LSPR technology and show how LSPR is useful for protein characterization and quantitation.

BASICS OF LSPR TECHNOLOGY

Unlike surface plasmon resonance (SPR), a sister technology that uses flat gold thin films surrounded by sophisticated instrumentation, LSPR technology resides in the sensor itself and requires only elementary components.7,8 An LSPR instrument schematic is shown in Figure 1A. White light from a tungsten halogen bulb is directed onto the nanostructured LSPR film. The light interacts with the localized surface plasmons so some wavelengths are absorbed by the film. The reflected light therefore has certain wavelengths missing and is analyzed by a spectrometer. Figure 1B compares the absorptions of a gold nanostuctured LSPR film (red) and a regular gold thin film (dark yellow). The insets are camera images of both films. The regular gold thin film absorption is featureless and can be described by Rayleigh scattering (~1/λ, in which λ is the wavelength of light).4 In contrast, the nanostructured LSPR film exhibits strong absorption near 550 nm that is the linear superposition of Rayleigh scattering and localized surface plasmon absorption. The LSPR has a natural width of ~80–100 nm, but its maximum position, or λmax, can be tracked in real time with a resolution of a few picometers (Figure 1C).

Figure 1. Description of the LSPR technology. A) An instrument is composed of a source of white light, the LSPR film, and a spectrometer to read the reflected light. For convenience, in this configuration of an instrument, the light arrives at 90° through optical fibers. The reflected light is sent to the linear array CCD spectrometer (Ocean Optics, USB 2000, with SONY 2048 pixel chip that cover 430–730 nm range) using optical fibers for analysis. B) Absorption spectra of a regular flat gold surface (dark yellow) and the LSPR nanostructured film (red), along with a true color of both films. Absorption is related to the reflected signal through Abs = –log10 (ref/normalization). The peak position λmax is computed in real time by a proprietary algorithm. With current hardware, the standard deviation on the peak position is ~6 pm. C) Sensorgram showing the binding and elution of human IgG on a Protein A surface in real time at 3 Hz. D) To test the robustness and reproducibility of the LSPR technology, the same experiment is performed 8 times on four surfaces simultaneously. By computing the shift of the IgG injection after 60 sec, the coefficient of variation (CV) of the 32 readings is ~2.1%.

The position of the LSPR absorption, λmax, is sensitive to the nature of the interface between the nanostructured gold surface and its environment. For example, in Figure 1C, the LSPR surface is used for the label-free and real time detection of a human IgG antibody binding to immobilized Protein A. Before injection of the antibody, the LSPR signal λmax is constant, thus Δλmax = 0. When the antibody is injected, the resonance starts to red-shift, and reaches 3,200 pm after 3 min. After a brief rinse with phosphate buffered saline (PBS), the solution pH is lowered to pH 2 so the interaction between the antibody and the Protein A surface is disrupted. This produces a sudden decrease of the LSPR signal indicating that the antibody has been removed from the surface. When the pH is re-established to its PBS value, the LSPR signal returns to its original value, i.e., Δλmax = 0, an indication that the sensor has been regenerated. Traces such as this one, also called sensorgrams, represent raw LSPR data. Unlike SPR, LSPR does not require correction for bulk effect signals that occur during injections and rinses. This is a result of the plasmons' localized nature and the fact that they extend and sense only 20–30 nm from the surface. Regular surface plasmons of thin films sense changes up to 200–1,000 nm away from the surface.7–8

To illustrate the performance of current LSPR technology, a 4-channel biochip is used to monitor channels simultaneously in real time when the same human antibody is injected. Figure 1D superimposes the sensorgrams when 8 cycles and binding-regeneration are performed. Analysis of reproducibility is performed by computing the shift in λmax after an arbitrary 60 sec time lapse after the injection. For these 32 independent repeats, the shift reading yields a coefficient of variation (CV) of ~2.1%. A CV of similar magnitude is measured across channels and across different sensors of the same batch. This underscores the apparent consistencies possible for both the manufacturing and biofunctionalization of the nanostructured films.

SELECTED APPLICATIONS

Label-free techniques are powerful tools in bioprocess monitoring for quality management applications and in R&D for applications such as antibody screening, epitope binning and mapping, and affinity and kinetic studies. LSPR technology fits into these categories based on recent published results.4–6,8 Concentration monitoring and kinetic analyses, two applications of LSPR technology, are discussed below.

The high degree of binding reproducibility traces can be used to evaluate dose response or the titer of a particular analyte. In Figure 2A, the response of a Protein A biosensor is measured when exposed to different concentrations of human IgG ranging from 7 μg/mL to 10 mg/mL. There are three overlapping repeats at 500 μg/mL. For quantitation, the LSPR sensor shift is read as a function of the analyte concentration at arbitrarily set check times. Figure 2B reports the reading after 25 and 120 sec for each concentration. The readings can be fit with a logistic model as shown by the dashed lines through the data points. This approach establishes a calibration curve for the sensor or batch of sensors, and is used for determining the test antibody concentration by comparing the shift produced by the test antibody with the calibration curve.

Figure 2. Examples to illustrate the depth of the LSPR detection. A) Dose-response of a Protein A surface for human IgG binding, ranging from ~7 μg/mL to 10 mg/mL. 60 μL of IgG is introduced at 30 μL/min to the LSPR surface, followed by a rinse of PBS. The green (25 sec) and blue (120 sec) arrows indicate reading time for generating the calibration curves in panel B. B) Shifts after 25 sec (green line) and 120 sec (blue line) are plotted against the IgG concentrations. The dashed line represents a fit to the logistic model and is used as a calibration curve. C) Illustration of the influence of the matrix on the performance of the LSPR sensor. The binding properties for a Protein A/IgG binding are measured for IgG spiked in PBS and in cell culture media of non-expressing cells at an identical concentration (performed at a customer site). There is a close relationship between the two media, indicating only a minimal perturbation of the media on the performance of the LSPR sensor. D) Illustration of the LSPR capability to compute kinetic parameters. The model used here has caboxybenzene sulfonamide (CBS) on the surface and bovine carbonic anhydrase II (CAII), a 29 kDa molecule, in solution at concentrations of 100, 33.3, 11.1, 3.33, and 1.11 μg/mL. The gray line is the response of CAII on a reference surface lacking CBS on the surface. Fits with Scrubber 2 software to yield a KD of ~1.5 μM, consistent with results reported using SPR techniques.9–10

Figure 2C compares the dose-response for human IgG measurements performed in PBS and crude media, and indicates only a marginal difference. In fact, LSPR technology has been used to quantify the amount of IgG expressed by Chinese hamster ovary cells and in media obtained from a fermenter within a few percent of the value obtained by HPLC. More generally, LSPR technology is compatible with various matrices, including crude media, whole blood, cell lysates, and other complex buffers containing up to 10% dimethyl sulfoxide (DMSO).

Similarly, LSPR technology is particularly useful for kinetic analyses because in injection and rinse steps there is negligible bulk effect, which minimizes the need for data manipulation, a potentially major source of variance. Figure 2D represents the kinetic data for the binding and dissociation of bovine carbonic anhydrase II, a 29 kDa protein, to immobilized sulfonamide ligands. Various repeats show that the responses can be reproduced, and allow the kinetic parameters to be computed using Scrubber 2 software, designed for data analysis and used for SPR. The rate constants (kon, koff, and KD) are compatible with values reported in the literature using SPR.9–10

FROM LABEL-FREE TO LABELED DETECTION FOR ENHANCING SENSITIVITY

During bioprocessing, detection of host-cell protein, Protein A amounts leached from purification columns, and absence of live mycoplasma are critical for batch validation. These tests currently are performed with methods orthogonal to label-free techniques, and often rely on ELISA assays performed by a third-party laboratory. While results are being determined, production is in a stand-by mode.

Although label-free LSPR does not require sample pretreatment, its sensitivity is restricted to the 10–200 ng/mL range of detection dependent on the protein being analyzed. To bridge the gap between label-free and labeled technology, LSPR detection can be enhanced with an enzyme-labeled protein capable of selectively binding to the antigen of interest, such as the low level contaminants mentioned above. With a single reading platform, it becomes possible to expand levels of detection into the pico- to femtogram range for an assay time <30 min. The principle of LSPR-labeled detection is illustrated in Figure 3A. An enzyme, such as alkaline phosphatase, is used to convert a substrate into an insoluble form. After deposition on the LSPR surface, the plasmon peak position red-shifts by between 20 and 100 nm (Figure 3B).

Figure 3. A) Illustration of the label-free and labeled assay implementation using an LSPR surface. In the labeled case, an enzyme is linked to a specific protein. Similar to an immunoassay, the enzyme is used to convert a soluble substrate into an insoluble product. As soon as the product deposits on the surface, the LSPR color changes. After 2 min of reaction with an estimated 2 nm of product deposited on the surface, the color of the surface becomes blue and corresponds to a shift of 50–100 nm with respect to the original plasmon. B) The proof that color change is specific to a LSPR surface becomes evident based on the comparison of LSPR and regular gold sensors for the detection of various amounts of alkaline phospahatase. Although LSPR sensors show a significant dose-response, the regular gold sensor shows minimal response. C) Use of label-free and labeled LSPR for the detection of residual Protein A (Pro A), in a background of 5 mg/mL IgG. In the left panel, residual Pro A is spiked in a solution of 5 mg/mL IgG; Pro A is detected label-free in real time to ~10 ng/mL or 2 ng/mg-IgG of Pro A; in the right panel, the sandwich antibody is labeled with alkaline phosphatase, and its plasmon is read. After addition of the substrate and 2 min of incubation, the LSPR array is read again to screen for a positive response. The graphs on the right represent a control surface exposed to BSA (lacking exposure to Pro A) and a surface exposed to 10 pg/mL or 2 pg/mg of IgG of Pro A. Spectroscopically, the positive surface displays a 44 nm shift, whereas the control shifts by only 6 nm.

The color shift is caused by insoluble substrate being deposited onto the sensor surface, whereby the nanostructured surface acts as a transducer. The presence, not the color, of the precipitate is measured. For example, the reduction rate of nitro-blue tetrazolium (NBT) by alkaline phosphatase (0.002–0.007 OD/min)11 corresponds to a deposit on the surface of 2–5 nm/min. On the other hand, the LSPR surface can detect <0.1 nm NBT deposition, because that amount would cause a measurable index of refraction change at the surface of ~10–3.8 This is why detection is faster than the traditional ELISA when the same type of experiment is performed on the LSPR surface.

The particularities of the nanostructured LSPR surface are illustrated in Figure 3B. Antigen and alkaline phosphatase have been immobilized in parallel on both the LSPR and regular gold surfaces. After 2 min of substrate conversion, there is a minimal change on the regular gold chip at an even higher antigen dose, while clear and distinct coloration appears on the LSPR chip. A real case scenario for the detection of residual Protein A, in a 5 mg/mL IgG background, was undertaken to demonstrate the feasibility of this approach. Although the label-free detection can detect down to ~10 ng/mL of Protein A in a solution with 5 mg/mL IgG (Figure 3C, left), the labeled method was able to detect down to ~10 pg/mL of Protein A. The labeled-approach currently is a semiquantitative end-point assay, but there are indications that it can be recast into a real time quantitative assay as the technology advances.

CONCLUSIONS

Monitoring through LSPR requires only a white light source and a spectrometer. The platform also is largely insensitive to temperature and ultra-precise alignment and therefore can be assembled at a fraction of the cost of conventional SPR. Because of its engineering simplicity, LSPR is amenable to miniaturization for certain point-of-use assays. Academic laboratories already have reported palm-size prototypes based on LSPR.12 The emergence of a commercial LSPR platform that reads the absorption of white light by a nanostructured thin film holds great promise for basic protein science and for applications such as quality control and quality assurance. It permits label-free quantitation of biomolecules at the ng/mL level, yet makes contaminant detection in solution at femtogram to picogram levels possible, using the addition of labels that provide notable improvements over current ELISA testing by enhancing the sensitivity and speed of assay responses. LSPR technology is equivalent to current state-of-the art SPR instruments in terms of data quality, sensitivity, and dynamic range, while also being scalable relative to throughput. In its current inception, the LSPR instrument can read eight channels simultaneously with future modules enabling a greater number of channels.

Daniele Gerion and Gwo-Jen Day are both senior scientists at LamdaGen Corporation, Menlo Park, CA, 650.235.5971, dgerion@lamdagen.com

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