Measurement of biomarker proteins for point-of-care early detection and monitoring of cancer

Abstract

This critical review evaluates progress toward viable point-of-care protein biomarker measurements for cancer detection and diagnostics. The ability to measure panels of specific, selective cancer biomarker proteins in physicians' surgeries and clinics has the potential to revolutionize cancer detection, monitoring, and therapy. The dream envisions reliable, cheap, automated, technically undemanding devices that can analyze a patient's serum or saliva in a clinical setting, allowing on-the-spot diagnosis. Existing commercial products for protein assays are reliable in laboratory settings, but have limitations for point-of-care applications. A number of ultrasensitive immunosensors and some arrays have been developed, many based on nanotechnology. Multilabel detection coupled with high capture molecule density in immunosensors and arrays seems to be capable of detecting a wide range of protein concentrations with sensitivity ranging into the sub pg mL(-1) level. Multilabel arrays can be designed to detect both high and ultralow abundance proteins in the same sample. However, only a few of the newer ultrasensitive methods have been evaluated with real patient samples, which is key to establishing clinical sensitivity and selectivity.

Fig. 1

Example of an immunoarray strategy designed to detect cancer biomarker proteins (PSA = prostate specific antigen). Gold nanoparticles providing high spot areas are linked to primary antibodies that capture the protein analytes. After washing, a labeled secondary antibody, or as illustrated here a multi-labeled nanoparticle with attached secondary antibody, is added. This species binds to another site on the captured analyte molecules. After additional washing with blocking agents to remove non-specific binding of the labeled species, electrical or optical detection is used to “count” the number of bound labels that is proportional to protein analyte concentration.

Fig. 2

Illustration of SPR arrays on gold films that can be used for measuring multiple protein biomarkers shown as a side view of an array that is coupled to a CCD camera for imaging. On the right is the top view of a 5 × 5 array.

Fig. 3

Amplification strategies for electrochemical immunosensors using nanoparticles or other moieties attached to secondary antibody Ab₂.

Fig. 4

Tapping mode atomic force microscopy (AFM) images of: (a) SWNT forest on smooth silicon and (b) anti-biotin antibody functionalized SWNT on smooth silicon. (c) PDDA/gold (5 nm) nanoparticle bilayer on a smooth mica surface suggesting a densely packed nanoparticle layer; (d) phase contrast image of gold nanoparticle electrode on mica, again suggestive of full coverage with the nanoparticles; and (e) gold nanoparticle electrode as in “c” after covalent linkage of the antibody anti-PSA onto the surface. Images a and b reproduced with permission from X. Yu, S. Kim, F. Papadimitrakopoulos, J. F. Rusling, Mol. BioSyst., 2005, 1, 70–78. Copyright Royal Society of Chemistry (UK), 2005. Images c, d, and e reproduced with permission from ref. , Copyright American Chemical Society, 2009.

Fig. 5

Amperometric response for nanostructured immunosensors incubated with PSA in 10 μL serum in buffer containing 1 mM hydroquinone after injecting 0.04 mM H₂O₂ to develop the signal (A) current for SWNT immunosensors at −0.3 V and 3000 rpm using the Ab₂–CNT–HRP bioconjugate with 170 labels per 100 nm at PSA concentrations shown: controls: (a) full SWNT immunosensor omitting addition of PSA, (b) immunosensor built on bare PG surface for 100 pg mL⁻¹ PSA, (c) immunosensor built on Nafion–iron oxide-coated PG electrode for 100 pg mL⁻¹ PSA. (B) Influence of PSA concentration on steady state current for SWNT immunosensor using Ab₂–CNT–HRP bioconjugate. (C) Current at −0.3 V and 3000 rpm for AuNP immunosensors using Ab₂–magnetic bead–HRP with 7500 labels/bead at PSA concentrations shown. Controls: (a) immunosensors built on bare PG at 10 pg mL⁻¹ PSA; (b) immunosensors built on PDDA coated PG surface at 10 pg mL⁻¹ PSA; (D) influence of PSA concentration on steady state current for AuNP immunosensor using multi-label Ab₂–magnetic bead–HRP. A and B reproduced with permission from ref. , copyright American Chemical Society 2006. C and D reproduced with permission from ref. , Copyright American Chemical Society, 2009.