Optical protein sensor for detecting cancer markers in saliva

Abstract

A surface immobilized optical protein sensor has been utilized to detect Interleukin-8 (IL-8) protein, an oral cancer marker, and can reach limit of detection (LOD) at 1.1 pM in buffer without using enzymatic amplification. Only after applying enzymatic amplification to increase the signal level by a few orders of magnitude, ELISA can reach the LOD of 1 pM level. We then develop the confocal optics based sensor for further reducing the optical noise and can extend the LOD of the surface immobilized optical protein sensor two orders in magnitude. These improvements have allowed us to detect IL-8 protein at 4.0 fM in buffer. In addition, these sensitive LODs were achieved without the use of enzymatic signal amplification, such that the simplified protocol can further facilitate the development of point-of-care devices.

The ultra sensitive optical protein sensor presented in this paper has a wide number of applications in disease diagnoses. Measurements for detecting biomarkers in clinical sample are much more challenging than the measurements in buffer, due to high background noise contributed by large collections of non-target molecules. We used clinical saliva samples to validate the functionality of the optical protein sensor. Clinical detection of disease-specific biomarkers in saliva offers a non-invasive, alternative approach to using blood or urine. Currently, the main challenge of using saliva as a diagnostic fluid is its inherently low concentration of biomarkers. We compare the measurements of 40 saliva samples; half from oral cancer patients and half from a control group. The data measured by the optical protein sensor is compared with the traditional Enzyme-Linked Immunosorbant Assay (ELISA) values to validate the accuracy of our system. These positive results enable us to proceed to using confocal optical protein sensor to detect other biomarkers, which have much lower concentrations.

Introduction

ELISA is a standard technique to quantify the amount of protein in a solution. Protein targets are immobilized on a surface and labeled with an enzyme that can continually produce a colored product which accumulates in the solution for detection. If we track the information pathway, we find that the signal originates in the fluid sample volume, moves to the surface and returns to the volume to be finally detected. Although the signal is amplified by an enzymatic reaction, the method for taking light absorbance measurements (fluid opacity) is a relatively insensitive principle compared to optical fluorescence methods (Wells, 2006, Janasek et al., 2006).

At low target concentrations, surface immobilization (Britland et al., 1992, Mooney et al., 1996, Lahiri et al., 1999, MacBeath and Schreiber, 2000) is an efficient detection principle, especially when the surface-to-volume ratio is increased (Khandurina and Guttman, 2002, Luo et al., 2005). In this work, we examine the effects of eliminating the enzymatic signal amplification and instead, directly labeling the immobilized proteins with fluorescent probes (Espina et al., 2004, Bashir, 2004). We increase the sensitivity of detection by using fluorescence optics to measure the intensity signal (Craighead, 2006) and reducing the sample volume for increased surface-to-volume ratio. The low sample volume requirement (tens of microliters) is advantageous for clinical situations in which only a small amount of the biological fluid is available for assay.

Detecting biomarkers from a 3D volume to a 2D surface needs smaller sample volumes and can improve LOD. The challenge of this approach is the non-specific binding of non-target molecules or fluorescent probes to the surface, both of which will affect the signal integrity. Our previous development of an ultra-sensitive electrochemical RNA/DNA biosensor (Gau et al., 2001) demonstrated that a well-controlled sensor surface could reduce the background noise due to non-specific molecular binding. With this optimized surface modification protocol, detection sensitivity much less than femtomolar concentrations was achieved. This achievement effectively leads to the elimination of PCR amplification of DNA/RNA targets which is usually needed to enable detection (Wang et al., 2005). Adopting a similar surface modification approach, we have shown in this paper that IL-8 can be detected at 1.1 pM level by using a surface immobilized sandwich assay technique. In comparison, the commercial IL-8 ELISA assay has a similar LOD at 1 pM; thus, we are able to achieve the same sensitivity but without the use of enzymatic signal amplification.

IL-8 has a molecular weight of 8.5 kDa and has clinical significance for oral cancer diagnosis (St John et al., 2004). Oral cancer, the fifth most common cancer in the United States, makes up for the largest number of cancers in the head and neck category. According to the American Cancer Society, approximately 34,000 people in the United States will be diagnosed with oral cancer in 2007. The survival rate of oral cancer is 60–80% when detected during its early stages; however, this number drops to 30–40% when the cancer is diagnosed during the advanced stages (Franzmann et al., 2005). Identifying molecular markers of early disease can aid in early diagnosis, which can improve patient prognosis (Sidransky, 2002). IL-8, as a salivary biomarker for early stage oral squamous cell carcinoma (OSCC), was discovered through a previous tissue-based expression profiling effort (St John et al., 2004). Among all of the body fluids that can be tested in the clinic, saliva is the easiest to access and its collection is the least invasive to the patient. In this effort, we first use a commercial ELISA kit to execute endpoint detection of the OSCC protein biomarker from a patient's saliva; it was found that IL-8 was significantly elevated in the saliva of oral cancer patients. It was highly discriminatory in detecting oral cancer by saliva analysis. The average level of IL-8 in cancer and control patients depends on several variables, such as the number of the subjects and their physical condition. Regardless of these variables, there is an overall increase in the average measurements of IL-8 in cancer patients (Wong, 2006, Rhodus et al., 2004).

Being a filtrate of the serum, saliva has been found to have an abundance of biomarkers with major clinical significance (Herath, 2003, Sonmezoglu et al., 2005). The biomarker concentration in whole saliva can be lower than that of the serum, as in the case of TNF-α, which is another protein biomarker for oral cancer. The salivary TNF-α concentration is approximately 30 pg/ml (2 pM) in oral cancer patients and 3 pg/ml (176 fM) in healthy individuals. For detecting TNF-α in saliva, the LOD, a measure of the signal-to-noise ratio, must be several orders of magnitude lower than the baseline concentration. A need of more sensitive protein sensor is obvious. At low target concentrations, the signal is usually dominated by the background noise, which is caused by non-specific binding and optical noise. We have minimized the non-specific binding by surface modification. In optical system, the optical noise can be reduced through the utilization of confocal optics (Shotton, 1989). The use of confocal optics allows us to confine the detection volume by allowing the signal to pass through a pinhole, thereby rejecting signals that are not from the focal plane of the microscope. Indeed, a LOD as low as 4.0 fM was achieved. This value is approximately 200 times lower than the LOD for surface immobilized techniques, which lack the confocal arrangement. Our confocal optics-based surface immobilized protein sensor is essentially an ultra-sensitive protein sensor, which has potential for a wide array of applications in disease diagnostics.

In this study, three advances in optical protein biosensor are presented: (1) without enzymatic signal amplification, the surface immobilized optical protein sensor can reach a LOD of detecting IL-8 at 1.1 pM in buffer, (2) with the utilization of confocal optics, the LOD of this sensor can be further reduced by two orders of magnitude (Schweitzer et al., 2002, Zajac et al., 2007), detecting IL-8 at 4.0 fM in buffer and (3) the optical protein sensor is validated through detecting IL-8 protein in clinical saliva samples by comparing with traditional ELISA measurements. Our results show that the patients with oral cancer can be clearly distinguished from the control subjects.