The nanosensensor technology described here is based on magnetic nanoparticle conjugates, which form nanoclusters on target interaction. The nanocluster formation (typically 300–500 nm) causes a rapid decrease in the water T2 relaxation time [13
]. Because of the inherently built-in amplification (each cluster formation affects billions of surrounding water molecules), the ability to sense in turbid samples and the ability to design different assay configurations (DNA, RNA, protein, metabolites), this method provides unique advantages. Water relaxation can be readily detected by benchtop relaxometers [23
] chemical nuclear magnetic resonance (NMR) systems, or even magnetic resonance imaging systems [17,24
]. In this study, we evaluated whether parallel measurements could be made to determine protein levels and protein activity in biologic samples using the same readout, the proportional decrease in T2 (δT2). As a clinically relevant model system, we used telomerase, a protein implicated in tumorigenesis, whose functional state is affected differently by a number of drugs currently under development [25
]. We show that the developed technology can quickly and accurately determine the amount of telomerase protein and assess for its levels of activity and activation.
High levels of hTERT mRNA have been observed in most tumors with high telomerase activity [26
], suggesting that hTERT is by itself an up-regulating factor in its own activation, essential in oncogenesis, tumor progression, and tumor invasion [27
]. However, some lesions and normal tissue with low or undetectable telomerase activity have been found to contain significant levels of hTERT mRNA, presumably due to either alternate splicing [28
] or posttranslational modifications such as phosphorylation-dephosphorylation [17,29–31
] that might alter the activity of the enzyme. Most importantly, high levels of telomerase activity have been reported in aggressive tumors and in metastatic lesions presumably due to up-regulation of telomerase reverse transcriptase [32
]. However, the effect of phosphorylation on the telomerase activity of these metastatic lesions is not well understood at this time.
On the basis of our results, the measured activity of telomerase has two main components: 1) the contribution from the total amount of telomerase protein and 2) the level of phosphorylation of telomerase protein. When both of these parameters are taken into account by multiplying the detected amount of telomerase protein (as the δT2 protein) with the amount of phosphorylation-induced activation of telomerase (δT2 activation) and plotted against the measured telomerase activity (δT2 activity), a linear correlation was observed (). This observation has important implications for the evaluation of novel antitelomerase therapies because both the level of activation and the amount of hTERT protein must be taken into account. It is thus highly advisable to determine not only the activity but also the amount of protein to determine whether a given telomerase activity level is either due to highly activated telomerase present in small amounts or to large amounts of less active enzyme, because this could have important implications for an antitelomerasegeared therapy.
In summary, we have developed an integrated nanosensor technology to determine the levels of a neoplastic protein marker (telomerase) and its level of activation in various cancer cells lines. With this method, results can be obtained more quickly, thereby using the same nanoparticle technology platform, instead of laborious and costly methods such as reverse transcription-PCR and Western blots. The method can be easily adapted to study other cancer-related protein markers such as metalloproteinases and caspases, among others. Current developments in NMR miniaturization [33
] and portable relaxometers [34
] will facilitate the implementation of the described technique in future cancer diagnostics, prognostics, and the assessment of antitelomerase-geared therapies.