Switch-based biosensors: a new approach towards real-time, in vivo molecular detection

Abstract

Although the ability to monitor specific molecules in vivo in real-time could revolutionize many aspects of healthcare, the technological challenges that stand in the way of reaching this goal are considerable and are poorly met by most existing analytical approaches. Nature, however, has already solved the problem of real-time molecular detection in complex media by employing biomolecular "switches". That is, protein and nucleic acids that sense chemical cues and, by undergoing specific, binding-induced conformational changes, transduce this recognition into high-gain signal outputs. Here, we argue that devices that employ such switches represent a promising route towards versatile, real-time molecular monitoring in vivo.

Figure 1

Recent years have seen the development of a large number of optical and electrochemical biosensors based on binding-induced conformational switching. Examples include: (a) A large family of optical sensors is based on the binding-induced conformational changes in proteins in the periplasmic binding protein family; this motion, in turn, modulates the emission of an attached fluorophore or fluorophores. Reproduced with permission from Ref. [15]. (b) Molecular beacons, which are stem-loop DNA molecules, open, and thus fluoresce, upon hybridization to a complementary target oligonucleotide. Reproduced with permission from Ref. [40]. (c) Electrochemical aptamer-beacons, undergo binding-induced “folding” of specific RNA or DNA aptamers, which can be monitored electrochemically via changes in electron transfer from an attached redox tag to a supporting electrode.

Figure 1

Recent years have seen the development of a large number of optical and electrochemical biosensors based on binding-induced conformational switching. Examples include: (a) A large family of optical sensors is based on the binding-induced conformational changes in proteins in the periplasmic binding protein family; this motion, in turn, modulates the emission of an attached fluorophore or fluorophores. Reproduced with permission from Ref. [15]. (b) Molecular beacons, which are stem-loop DNA molecules, open, and thus fluoresce, upon hybridization to a complementary target oligonucleotide. Reproduced with permission from Ref. [40]. (c) Electrochemical aptamer-beacons, undergo binding-induced “folding” of specific RNA or DNA aptamers, which can be monitored electrochemically via changes in electron transfer from an attached redox tag to a supporting electrode.

Figure 1

Recent years have seen the development of a large number of optical and electrochemical biosensors based on binding-induced conformational switching. Examples include: (a) A large family of optical sensors is based on the binding-induced conformational changes in proteins in the periplasmic binding protein family; this motion, in turn, modulates the emission of an attached fluorophore or fluorophores. Reproduced with permission from Ref. [15]. (b) Molecular beacons, which are stem-loop DNA molecules, open, and thus fluoresce, upon hybridization to a complementary target oligonucleotide. Reproduced with permission from Ref. [40]. (c) Electrochemical aptamer-beacons, undergo binding-induced “folding” of specific RNA or DNA aptamers, which can be monitored electrochemically via changes in electron transfer from an attached redox tag to a supporting electrode.

Figure 2

Switch-based sensors are finding increasing application in intracellular studies. (a) A maltose sensor based on the binding-induced switching of maltose-binding protein (a schematic of which is shown in Figure 1a) supports rapid, intracellular measurements of the sugar. The optical reporter groups are two florescent protein variants that are fused to the switching domain and that serve as a RET pair. As all of the components of this system are contained within a single polypeptide chain, this sensor can be expressed in situ. Reproduced with permission from Ref. [27]. (b) Molecular beacons (schematic shown in Figure 1b), which support rapid fluorescence detection of specific oligonucleotide sequences, have been employed to localize and quantify endogenous mRNA levels within living cells, including the detection of mRNA encoding the protein survivin in live human dermal fibroblast cells. In this example, the molecular beacon is delivered into the cell by coupling it with the TAT polypeptide, a signaling sequence that leads to rapid and efficient internalization.

Figure 2

Switch-based sensors are finding increasing application in intracellular studies. (a) A maltose sensor based on the binding-induced switching of maltose-binding protein (a schematic of which is shown in Figure 1a) supports rapid, intracellular measurements of the sugar. The optical reporter groups are two florescent protein variants that are fused to the switching domain and that serve as a RET pair. As all of the components of this system are contained within a single polypeptide chain, this sensor can be expressed in situ. Reproduced with permission from Ref. [27]. (b) Molecular beacons (schematic shown in Figure 1b), which support rapid fluorescence detection of specific oligonucleotide sequences, have been employed to localize and quantify endogenous mRNA levels within living cells, including the detection of mRNA encoding the protein survivin in live human dermal fibroblast cells. In this example, the molecular beacon is delivered into the cell by coupling it with the TAT polypeptide, a signaling sequence that leads to rapid and efficient internalization.

Figure 3

An E-AB sensor for the detection of cocaine (schematic shown in Figure 1c) supports real-time monitoring of cocaine directly in undiluted blood serum as it flows through a sub-microliter microfluidic chamber. (a) The MECAS chip incorporates three electrodes: “R” is the reference electrode, which provides a standard by which voltages can be accurately measured; “W” is the “working electrode”, which interrogates the attached aptamer and is the heart of the biosensor; “C” is the counter-electrode, which completes the circuit. (b) The system continuously monitors the presence of cocaine. When challenged with cocaine in serum (10, 50 and 100 µM), the current (I_p) increases before dropping back to baseline after washing with cocaine-free serum. Figure adapted with permission from Ref. [37].