A novel optical biosensor based on long-range surface plasmon-polariton (LRSPP) waveguides is demonstrated for the detection of leukemia markers in patient serum using a functionalization strategy based on Protein G. The sensor consists of thin straight Au waveguides (5 μm × 35 nm × 3.2 mm) embedded in fluoropolymer CYTOP™ with a fluidic channel etched into the top cladding. B-cell leukemia is characterized by a high B-cell count and abnormal distribution of immunoglobulin G kappa (IgGκ) and lambda (IgGλ) light chains in serum. The detection of leukemic abnormalities in serum was performed based on determining IgGκ-to-IgGλ ratios (κ : λ). Three patient sera were tested: high kappa (HKS, κ : λ ~12.7 : 1), high lambda (HLS, λ : κ ~6.9 : 1) and normal (control) sera (NS, κ : λ ~1.7 : 1). Au waveguides were functionalized with Protein G and two complementary immobilization approaches were investigated: a) the reverse approach, where the Protein G surface is functionalized with patient serum and then tested against goat anti-human IgG light chains in buffer, and b) the direct approach, where the Protein G surface is functionalized with goat anti-human IgGs first and then tested against patient serum. The reverse approach was found to be more effective and robust because Protein G-functionalized surface performs as an "immunological filter" by capturing primarily IgGs out of the pool of serum proteins. For the reverse approach, the ratios measured were 3.7 : 1(κ : λ), 9.7 : 1(λ : κ) and 1.9 : 1(κ : λ) for HKS, HLS and NS, respectively, which compare favorably with corresponding protein densitometry measurements. The respective ratios for the direct approach were 2.6 : 1(κ : λ), 2.6 : 1(λ : κ) and 1.7 : 1(κ : λ). The binding strength and cross-reactivity of goat anti-human IgGs light chains were also determined using pure solutions. The LRSPP biosensor along with the innovative "reverse approach" can provide a low-cost and compact solution to B-cell leukemia screening.
PCR clonality with direct cell lysis of CSF is feasible, and it may overcome the limitation of DNA isolation. This PCR method may be particularly useful for small volume and low cell CSF when flow cytometry is inconclusive.
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