ARTICLES b 1.800 p-type nanowire(NWl, Fig 2a)and n-type nanowire (NW2, Fig. 2a) devices after M Nw1 introduction of 0.9 ng/ml of PSA showed a conductance increase in Nwl and a con- ductance decrease in Nw2, whereas the con- ductance returned to the baseline value of each device after introduction of buffer solu- tion without PSA. The magnitudes of the inductance changes in the two devices 1,00020003,0004,0005,000 were nearly the same and consistent with the concentration-dependent conductance Figure 2 Multiplexed detection with nanowire arrays (a)Complementary sensing of PSA using p-type measurements(Fig. 1). Similar behavior (NW1)and n-type(NW2)silicon- nanowire devices in the same array. The vertical solid lines observed with different concentrations o times at which PSA solutions of (1)0.9 ng/ml, (2)0.9 ng/ml, (3)9 pg/ml, (4)0.9 pg/ml and of PSA(Fig. 2a); that is, the p- and n-type E(5)5 ng/ml were connected to the microfluidic channel (b) Conductance-versus time data recorded devices showed ncentration E simultaneously from two silicon-nanowire devices in an array, where Nwl was functionalized increases and decreases, respectively, in con- s solutions of(1)9 pg/ml PSA, (2)1 pg/ml PSA, (3)10 Hg/ml BSA, (4) a mixture of i ng/ml PSA and ductance when the solution was alternated between psa and buffer a the points where the solution flow was switched from protein to pure buffer solutions e These experiments demonstrate key points bout multiplexed electrical de bserved for the or 2 fM. Similar ultrasensitive detection was achieved in studies and n-type elements are consistent with specific binding of PSa to e of carcinoembryonic antigen(CEA), 100 fg/ml or 0.55 fM,and field-effect devices, since the negatively charged protein will cause e mucin-1, 75 fg/ml or 0.49 fM,(see Supplementary Fig. 2 online) accumulation and depletion of charge carriers in the p-type and o using silicon-nanowire devices modified with mAbs for CEA and n-type nanowire elements, respectively. Second, the complementary tively electrical signals from p-and n-type devices provide a simple yet a in competitive binding experiments with bovine serum albumin noise or nonspecific binding of protein to one device; that is, real and 2(BSA)(Fig. Id). Conductance-versus-time measurements recorded selective binding events must show complementary responses in the on a silicon-nanowire device modified with Abl showed similar p-and n-type devices. The presence of correlated conductance signals onductance changes as above when 9 and 0.9 pg/ml solutions of in both devices(Fig. 2a), which occur at points when buffer and PSA were delivered to the device. These results show that reproducible PSA/buffer solutions are changed, illustrates clearly how this multi device-to-device sensitivity was achieved. Moreover, delivery of a plexing capability can be used to distinguish unambiguously nois solution containing 0.9 Pg/ml PSA and 10 ug/ml BSA showed the from protein-binding signals. conductance increase as a solution containing only PSA this concentration, whereas no conductance change was observed en the Bsa solution alone was delivered. These latter data demon- strate excellent selectivity and also show that high sensitivity was not Table I Summary of multiplexed detection experiments using mAb rec lost even with a 10-million-fold higher concentration of other proteins (f-PSA), Abl and cross-reactive for f-PSA and PSA-ACT complex, Ab2 We investigated the devices' modification chemistry to define their Conductance change, ns sensitivity limits. Atomic force microscopy measurements of the initial aldehyde-silane layer thickness on single nanowires(Fig. le)showed a Protein sample NWI-Abl NW2-Ab2 systematic increase with modification time. This thickness increase is onsistent with previous studies showing that similar silane reagents f-psA 154 can form multila ers25,26. Measurements of sensitivity showed that the sensor response decreases rapidly for initial reaction times >30 min The observed decrease in sensitivity is consistent with expectations for f-PSA a field-effect sensing device, and moreover, shows that the surface psa-Act modification chemistry must be controlled to achieve reproducible psa-act high-sensitivity devices. PSA-ACT PSA-ACT 55DDDDD Multiplexed detection with nanowire arrays f-PsA, PSA-ACT,Ab1850,3,200,1×10 For our initial studies of multiplexed detection, we used an array f-PSA, PSA-ACT, Abl 8.5, 320, 1 x 107 containing both p-type and n-type silicon-nanowire devices modified f-PSA, PSA-ACT, Abl 0.85, 3, 200, 1 x 107 138 with Abl. The incorporation of p-and n-type nanowires in a f-PSA, PSA-ACT, Abl 850, 0.32, 1 x 107 ingle sensor chip enables discrimination of possible electrical cross- f-PSA, Abl talk and/or false-positive signals by correlating the response versus ND me from the two types of device elements. Notably, simultaneous conductance changes are shown in Supplementary Figure 3onmesed to obtain VOLUME 23 NUMBER 10 OCTOBER 2005 NATURE BIOTECHNOLOGYor B2 fM. Similar ultrasensitive detection was achieved in studies of carcinoembryonic antigen (CEA), 100 fg/ml or 0.55 fM, and mucin-1, 75 fg/ml or 0.49 fM, (see Supplementary Fig. 2 online) using silicon-nanowire devices modified with mAbs for CEA and mucin-1, respectively. We further investigated the devices’ reproducibility and selectivity in competitive binding experiments with bovine serum albumin (BSA) (Fig. 1d). Conductance-versus-time measurements recorded on a silicon-nanowire device modified with Ab1 showed similar conductance changes as above when 9 and 0.9 pg/ml solutions of PSA were delivered to the device. These results show that reproducible device-to-device sensitivity was achieved. Moreover, delivery of a solution containing 0.9 pg/ml PSA and 10 mg/ml BSA showed the same conductance increase as a solution containing only PSA at this concentration, whereas no conductance change was observed when the BSA solution alone was delivered. These latter data demon￾strate excellent selectivity and also show that high sensitivity was not lost even with a 10-million-fold higher concentration of other proteins in solution. We investigated the devices’ modification chemistry to define their sensitivity limits. Atomic force microscopy measurements of the initial aldehyde-silane layer thickness on single nanowires (Fig. 1e) showed a systematic increase with modification time. This thickness increase is consistent with previous studies showing that similar silane reagents can form multilayers25,26. Measurements of sensitivity showed that the sensor response decreases rapidly for initial reaction times 430 min. The observed decrease in sensitivity is consistent with expectations for a field-effect sensing device27, and moreover, shows that the surface modification chemistry must be controlled to achieve reproducible high-sensitivity devices. Multiplexed detection with nanowire arrays For our initial studies of multiplexed detection, we used an array containing both p-type and n-type silicon-nanowire devices modified with Ab1. The incorporation of p- and n-type nanowires in a single sensor chip enables discrimination of possible electrical cross￾talk and/or false-positive signals by correlating the response versus time from the two types of device elements. Notably, simultaneous conductance-versus-time data recorded from p-type nanowire (NW1, Fig. 2a) and n-type nanowire (NW2, Fig. 2a) devices after introduction of 0.9 ng/ml of PSA showed a conductance increase in NW1 and a con￾ductance decrease in NW2, whereas the con￾ductance returned to the baseline value of each device after introduction of buffer solu￾tion without PSA. The magnitudes of the conductance changes in the two devices were nearly the same and consistent with the concentration-dependent conductance measurements (Fig. 1). Similar behavior was observed with different concentrations of PSA (Fig. 2a); that is, the p- and n-type devices showed concentration-dependent increases and decreases, respectively, in con￾ductance when the solution was alternated between PSA and buffer. These experiments demonstrate key points about multiplexed electrical detection with nanowire devices. First, the complementary conductance changes observed for the p-type and n-type elements are consistent with specific binding of PSA to field-effect devices, since the negatively charged protein will cause accumulation and depletion of charge carriers in the p-type and n-type nanowire elements, respectively. Second, the complementary electrical signals from p- and n-type devices provide a simple yet robust means for detecting false-positive signals from either electrical noise or nonspecific binding of protein to one device; that is, real and selective binding events must show complementary responses in the p- and n-type devices. The presence of correlated conductance signals in both devices (Fig. 2a), which occur at points when buffer and PSA/buffer solutions are changed, illustrates clearly how this multi￾plexing capability can be used to distinguish unambiguously noise from protein-binding signals. 2,000 1,800 1,800 1,600 1,400 1,200 0 900 1,800 2,700 3,600 1,600 1 2 3 45 1 2 34 1,400 1,200 1,000 Conductance (nS) Conductance (nS) 1,000 2,000 Time (s) Time (s) 3,000 4,000 5,000 NW2 NW1 NW2 NW1 0 a b Figure 2 Multiplexed detection with nanowire arrays. (a) Complementary sensing of PSA using p-type (NW1) and n-type (NW2) silicon-nanowire devices in the same array. The vertical solid lines correspond to times at which PSA solutions of (1) 0.9 ng/ml, (2) 0.9 ng/ml, (3) 9 pg/ml, (4) 0.9 pg/ml and (5) 5 ng/ml were connected to the microfluidic channel. (b) Conductance-versus-time data recorded simultaneously from two p-type silicon-nanowire devices in an array, where NW1 was functionalized with PSA Ab1, and NW2 was modified with ethanolamine. The vertical lines correspond to times when solutions of (1) 9 pg/ml PSA, (2) 1 pg/ml PSA, (3) 10 mg/ml BSA, (4) a mixture of 1 ng/ml PSA and 10 mg/ml PSA Ab1 were connected to the microfluidic channel. Black arrows in a and b correspond to the points where the solution flow was switched from protein to pure buffer solutions. Table 1 Summary of multiplexed detection experiments using nanowire devices modified with mAb receptors specific for free PSA (f-PSA), Ab1 and cross-reactive for f-PSA and PSA-ACT complex, Ab2 Conductance change, nS Protein sample [Protein] pg/ml NW1-Ab1 NW2-Ab2 f-PSA 1,700 192 154 f-PSA 850 185 132 f-PSA 8.5 98 81 f-PSA 0.85 45 50 f-PSA 0.085 15 10 PSA-ACT 3,200 ND 143 PSA-ACT 320 ND 124 PSA-ACT 3.2 ND 67 PSA-ACT 0.32 ND 19 f-PSA, PSA-ACT, Ab1 850, 3,200, 1
107 ND 140 f-PSA, PSA-ACT, Ab1 8.5, 320, 1
107 ND 118 f-PSA, PSA-ACT, Ab1 0.85, 3,200, 1
107 ND 138 f-PSA, PSA-ACT, Ab1 850, 0.32, 1
107 ND 15 f-PSA, Ab1 850, 1
107 ND ND ND corresponds to no detected conductance change. Sensor data used to obtain conductance changes are shown in Supplementary Figure 3 online. 1296 VOLUME 23 NUMBER 10 OCTOBER 2005 NATURE BIOTECHNOLOGY ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology