. 2020 May 5;45(7):604–618. doi: 10.1016/j.tibs.2020.03.011

Table 1.

Highlights of Proteolytic Activity in Real-Time Biosensors and Their Features

Type of biosensor	Target protease	Method of detection	Limit of detection	Suitable for in vivo	Advantage	Drawback	Refs
Optical	MMP-14	FRET	ND	✔	Versatile	May have misleading results	[59]^a
	MMP-14		ND	✔	Spectral properties and brightness	↓ In-depth penetration imaging	[59]^a
	MMP-14		ND	✔	↓ Background	Genetic modification is needed	[60]
	Micro PA + MMP-9		ND	✔	Multiplexing measurements	↓ In-depth penetration imaging	[40]^a
	Caspase-9		ND	✔	↑ SNR	↑ Photobleaching	[39]^a
	Caspase-3/8/9		ND	✔	↓ False-positive interference		[41]^a
	Trypsin, chymotrypsin	Double FRET	1 nM	✘	Double validation	Limited by spectra overlapping	[56]^a
					Multiplexing measurements
					Can be used as logic gates
	MMP-2	FRET, NIR-II optical imaging	ND	✔	Versatile to use for several proteases	May have misleading results	[35]^a
					↑ Spatial resolution through deep tissues	Limited for long-term studies
					Photostability and brightness	Laborious manufacture
	Caspase-3/8/9	Bioluminescence resonance ET	12.5 pM	✔	Does not need an excitation light source	May have misleading results	[61]
					Easy manufacture	Conditioned by surrounding medium
					Compared with FRET, ↓ photobleaching and ↓ light scattering
	Caspase-3	Nanosurface ET	12.0 pM	✔	↑ Sensitivity	↓ In-depth penetration imaging	[37]
					↓ Photobleaching
					↑ SNR
	Trypsin	Surface-enhanced Raman spectroscopy (SERS)	8.6 nM	✘	Multiplexing measurements	Cannot be used for a wide range of proteases	[62]
						Complex equipment needed
						Complex manufacture of the nanodomes
	PSA	Nanoplasmonic resonator, Raman	6.0 pM	✘	↑ Sensitivity	Complex equipment needed	[63]
	PSA	Nanoplasmonic resonator, Raman	6.0 pM	✘	Multiplexing measurements	Complex equipment needed	[63]
	MMP-2	Multispectral photoacoustic imaging	ND	✔	Spatial resolution	↑ Scattering level	[64]
		Multispectral photoacoustic imaging	ND	✔	Optical contrast without ionizing radiation	↓ In-depth penetration imaging	[64]
		Photoacoustic imaging	0.52 ng/ml	✔	Noninvasive	↑ Scattering level	[65]
					↑ Depth imaging	↓ In-depth penetration imaging
					↑ Spatial resolution
	Caspase-3	Optical imaging of plasmon rulers	ND	✔	↓ Photobleaching	Complex equipment is needed	[36]^a
					Stable over time	Prone to interference
					↑ Enhanced signal intensity
	MMP-2/9	Evanescent wave spectroscopy	32 nM/256 nM	✘	Cheap design	Difficult to adapt	[66]
					Portable	↓ Sensitivity
					Constant measurements	↓ Detection limits
Combined techniques	Thrombin	NIR Fluorescence/ PET	ND	✔	↑ Spatial resolution		[67]
	Thrombin		ND	✔	↑ Sensitivity	Complex equipment is needed	[67]
	MMP-2/9/13		ND	✔	↑ Accurate results	Complexity	[45]
	MMP-2/9/13		ND	✔	Whole-body imaging		[45]
	Cathepsin B	NIR fluorescence/ CT	ND	✔	↑ Spatial resolution	↓ In depth	[42]
					↑ Spatial resolution	Some interference
					Rapid imaging
	MMP-2/9	FRET/single-photon emission CT (SPECT)	4.8 ng/ml	✔	Nonradioactive probe	Complex equipment is needed	[43]
	Caspase-3/7	FRET/PET	ND	✔	↑ Spatial resolution	Unstable PET probe	[44]
	MMP-2	FRET/ MRI	0.64 pM	✔	↑ Time/spatial resolution	Complex equipment is needed	[46]^a
					↑ In-depth imaging
					↑ SNR
Non-optical	MMP-9	IDAMs	10.0 pM to 10.0 nM	✘	↑ Versatility	Limited to ‘2D systems’	[49]^a
	MMP-9	IDAMs	10.0 pM to 10.0 nM	✘	Suitable for complex Cause/effect studies	Limited to ‘2D systems’	[49]^a
	HIV-1 protease	Nanopore	47.0 pM	✘	Stable over time	Steric hindrance	[47]^a
					Multiplexing	Specific equipment is required
					Unambiguous response	Limited to 2D systems
					↑ Sensitive
	Caspase-3/7	MRI	ND	✔	↑ Time/spatial resolution	Complex equipment is needed	[48]^a
					↑ In-depth imaging	Very expensive
					↑ SNR	Multiplexing can be difficult

These examples are detailed in the main text.