Academic laboratories have embraced localized surface plasmon resonance (LSPR) as the “new wave” of label-free technology (1). This technique is based on the ability of colloidal metal nanoparticles or nanostructured metallic films to absorb light in a narrow wavelength range. Metal nanostructures “sense” changes occurring at their surfaces by shifting the frequency of the light they absorb or reflect. As a consequence, a basic LSPR system requires only optical fibers, a source of white light, and a detector (1,2). The simplicity of LSPR instrumentation contrasts with its exquisite sensitivity. Binding events and functional activity of nucleases (3) and proteases (4) can be monitored and quantified in real time based on observation of a single 20-nm nanoparticle — probably one of the world's smallest biosensing supports (5).

Nanostructured metallic films, rather than isolated metal nanoparticles, enable commercial endeavors by their robust and reproducible nature. With recent advances in nanofabrication and characterization, stable metal films can be manufactured at a large and cost-effective scale on a wide range of surfaces (2). Such films retain their nanostructures and the physical properties of nanoparticles. As a result, LSPR technology is quickly moving from proof-of-principle experiments to commercialization.

Key features of the technology include its marginal bulk effect and compatibility with various matrices, including cell media and sera. In addition, LSPR can accommodate label-free and labeled implementations, with the latter pushing its detection limits into the femto- to picomolar range (2). Various approaches using the same core technology allow detection of biologics in concentrations spanning from trace contaminants to levels of therapeutic antibodies in fermentation broths and cell culture supernatants. Furthermore, LSPR assays can be performed either at line or in line. As a result, this is a platform particularly well suited for all stages of bioprocessing, from development to monitoring and validation.

PRODUCT FOCUS: ANTIBODIES



PROCESS FOCUS: PRODUCTION AND DOWNSTREAM PROCESSING, PRODUCT DEVELOPMENT)



Who Should READ: QA/QC, PROJECT MANAGERS, AND UPPER-LEVEL MANAGERS



KEYWORDS: BIOSIMILARS, IGGS, DOSE RESPONSE, ROBUSTNESS AND RUGGEDNESS, ANALYTICAL METHODS



LEVEL: INTERMEDIATE

Photos 1:

Photos 1: (Click to enlarge)

We recently published a review covering the basics of LSPR and its application to protein characterization and quantitation (2). Here, we present the LightPath system, an LSPR platform developed by LamdaGen Corporation, and discuss the fundamental features that make it highly desirable for use in bioprocessing.

System and Features

Use of LSPR technology in bioprocess applications requires a sensing surface, a sample delivery apparatus, and a computer with software capability to acquire data in real time. So LamdaGen has developed the LightPath system. The instrument measures a biochip consisting of nanostructured metallic films formatted in four-channel (Photo 1A) or eight-channel (Photos 1B and 1C) simultaneous monitoring arrays. A light source and spectrometer are dedicated for each channel. White light is directed to the sensing surface by optical fibers. Reflected signals are collected by additional optical fibers connected to those individual spectrometers. Customized software collects spectra at a rate of 3–5 Hz. The reflectivity of impinging white light is monitored in real-time for each channel independently and then converted to a plasmon peak. Although the plasmon is 80–100 nm wide, a proprietary algorithm determines the position of its peak with a resolution of 2–6 pm. The system computes changes in plasmon position over time (the sensorgram) and displays it in real time for each channel.

Four or eight individual biochip channels are monitored in parallel by static or flow mode. Static mode is extremely useful for quick screening and fast qualitative analysis. A small amount of sample (30–200 µL) is injected using a pipettor and pipette tips before the binding reaction occurs. Static-mode sensorgrams are qualitative because mass transport affects binding reaction kinetics. But when samples are delivered to sensor surfaces using a modular syringe pump unit, the resulting flow better controls the LSPR–fluid interface and minimizes the effects of mass transport.

In flow-mode configuration, binding kinetics are highly reproducible and can be modeled and quantified. As in high-performance liquid chromatography (HPLC), fluidics are used to inject samples into fixed-volume loops so that they can be carried to the LSPR channels by running buffer. Typical sample volumes in flow mode are 50–2,000 µL, depending on the loop size. With current flow cell geometries, we have empirically determined that 10–50 µL/min flow rates produce binding sensorgrams exhibiting diffusion-limited effects, whereas at >60 µL/min the binding is fully kinetic limited.