One of the most challenging goals in biosensor development for clinical applications is to create surfaces that can detect low concentrations of biomarkers in complex media such as human serum. These surfaces should also ideally possess low biofouling properties, high binding capacity, and high sensitivity. One possible way to obtain these characteristics is to build 3D polyelectrolyte structures.
To evaluate the performance of these 3D polyelectrolyte structures as biosensing surface material, analytical instrumentation is needed to transduce any biochemical reaction into a measurable signal. Surface plasmon resonance (SPR) is a label-free technique which is based on total internal reflection of light, and it is sensitive to any changes in refractive index at the sensing surface. Therefore, it can be used for opaque and complex media such as human serum. This blog intends to highlight the capability of SPR (P4SPR™, Affinité Instruments) to detect prostate-specific antigen (PSA) in human serum samples by using various types of 3D polyelectrolyte structures compared to their 2D counterpart. On the other hand, biolayer interferometry (BLI) failed to achieve high sensitivity in detecting PSA in undiluted human serum samples. This research is based on the paper by Pan and others titled “Three-dimensional biosensor surface based on novel thorns-like polyelectrolytes” .
3D Polyelectrolyte Structures - A Case Study
The research article by Pan et al. described the synthesis of 3D polyelectrolyte structure as a novel biosensing surface to detect clinical biomarkers such as prostate-specific antigen (PSA). The cationic polyelectrolyte backbone is based on poly-L-lysine (PLL) and was electrostatically adsorbed onto gold or glass surfaces . After rigorous structural characterization by surface characterization methods, the 3D-PETx structures at various grafting ratios (10, 30, 40%) of biotinylated-oligoethylene glycol (OEG-biotin) sidechains, as well as their 2D-PET counterpart, were compared for their binding capacity and extent of antifouling by using BLI. The differences in structure are depicted in Figure 1. The main results were that the 3D-PETx structures, compared to 2D-PET, led to a higher binding capacity of ligands such as IgG and a negligible level of non-specific adsorption by bovine serum albumin .
Figure 1. Differences in 3D and 2D polyelectrolyte brush structures as depicted in Pan et al. . OEG is oligoethylene glycol and PLL is poly-L-lysine.
Detection of PSA in Serum with SPR
Surface plasmon resonance (SPR) is an optical technique that is based on the total internal reflection of light, which is highly sensitive to changes in refractive index induced by specific binding interactions on the sensor surface. Therefore, complex samples such as serum can be used without compromising the quality of the signal, assuming that the sensor surface is effective in preventing non-specific adsorption from other proteins. In this case study, the researchers switched from BLI to Affinité's P4SPR™ because a stable signal could not be achieved with BLI unless the serum was diluted to 10% . It was deduced that the light was being scattered in an opaque and complex medium . On the contrary, SPR can handle such complex biological samples. Thus, SPR chips were coated with anti-PSA-modified 3D-PETx or 2D-PET (Fig. 2), and they were then introduced to PSA in undiluted human serum. The P4SPR generated clear sensorgrams, and the height of the sensorgrams (or change in resonance wavelength) correlated well with PSA concentrations. Furthermore, 3D-PETx surface outperformed the 2D-PET ones, as they were more sensitive especially in the lower PSA concentration range (<5 ng/mL). This result is especially promising as the total PSA concentration in serum in this range of 2.5 - 10 ng/mL is often used to diagnose prostate cancer [2, 3], with 4.0 ng/mL (as well as 2.5 ng/mL) of total PSA as being probable for prostate cancer , and a reading of 0.7 ng/mL as a level for healthy individuals . As a result, the authors concluded that the 3D-PETx possessed excellent antifouling properties and higher probe density, which made them perform better in serum samples.
Figure 2. Schematic diagram of anti-PSA-modified 3D-PETx immobilized on a gold sensor chip to detect PSA by SPR.
BLI is an optical-based technique similar to SPR in that the signal collected is also reflected light. However, instead of a wavelength shift due to a change in refractive index, the amount of specific binding at the end of a glass fiber tip would cause a proportionate change in interference pattern. Figure 3 further explains how BLI works.
Figure 3. A schematic diagram of how BLI works, for example, for the detection of PSA by a biosensing layer modified with anti-PSA. The resultant signal is generated from the difference in spectral patterns reflected from the two reflection surfaces.
In this case study, the performance of 3D-PETx vs. 2D-PET modified surfaces was mainly evaluated by BLI. However, instead of BLI, SPR can also be used to evaluate biosensor surfaces for their binding capacity and extent of non-specific adsorption. Most importantly, BLI could not provide a stable signal for the two polyelectrolyte surfaces that were exposed to serum samples unless the serum was diluted to 10%. Instead, Affinité's P4SPR was able to measure the sensitivity of the two polyelectrolyte surfaces in undiluted human serum, concluding that the 3D-PETx had better sensitivity than the 2D-PET surfaces, especially in PSA concentrations lower than 5 ng/mL, which is of clinical importance. Thus, the ability of Affinité's P4SPR in conjunction with these 3D-PETx surfaces to detect low concentrations of biomarkers such as PSA in human serum is extremely significant in the development of diagnostic tools for clinical applications.
The Affinité Advantage
Besides being able to detect PSA in human serum as this blog has highlighted, our P4SPR has been used to detect other clinically relevant biomarkers such as the SARS-CoV-2 anti-nucleocapsid antibodies in human serum, HER2 biomarker in cell lysate, and anti-asparaginase antibodies in human serum. These studies were all conducted by Prof. Jean-François Masson’s research group from the Université de Montréal.
Affinité Instruments’ P4SPR™ is a very user-friendly instrument to evaluate the performance of novel biosensing surfaces. The gold sensing surface can be tailored to accommodate all types of surface chemistries. In addition, samples do not need much sample preparation and can be directly injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.
Simplicity - Fast training, fast results
Versatility - Pharmaceutical, biosensing, assay development applications
Economy - Affordable, accessible
Wenwei Pan , Ziyu Han , Ye Chang , Xuexin Duan. “Three-dimensional biosensor surface based on novel thorns-like polyelectrolytes.” Biosens. Bioelectron. (2020) 167, 112504.
Pavel Damborsky, Martina Zamorova, and Jaroslav Katrlik. “Determining the binding affinities of prostate-specific antigen to lectins: SPR and microarray approaches.” Proteomics (2016) 16, 3096–3104.
J. Breault-Turcot, H.-P. Poirier-Richard, M. Couture, D. Pelechacz and J.-F. Masson. “Single chip SPR and fluorescent ELISA assay of prostate specific antigen.” Lab Chip (2015) 15, 4433.
Gizem Ertürk, Haluk Özen, M. Askın Tümer, Bo Mattiasson, Adil Denizli. “Microcontact imprinting based surface plasmon resonance (SPR) biosensor for real-time and ultrasensitive detection of prostate specific antigen (PSA) from clinical samples.” Sens. Actuators B Chem (2016) 224, 823–832.
M.H. Jazayeri, H. Amani, A.A. Pourfatollah, A. Avan , G.A. Ferns, and H. Pazoki-Toroudi. “Enhanced detection sensitivity of prostate-specific antigen via PSA-conjugated gold nanoparticles based on localized surface plasmon resonance: GNP-coated anti-PSA/LSPR as a novel approach for the identification of prostate anomalies.” Cancer Gene Ther. (2016) 23, 365–369.