ARTICLES Figure 4 Sch (Top)Silicon-nanowire devices modified with oligonucleotide primer. (Middle)A solution containing telomerase is delivered to the device array. and telomerase binds in a concentration-dependent process. Bottom) Subsequent addition of dNTPs leads to telomerase-catalyzed primer extension/elongation. showed little change in baseline for either NWl or NW2, although subsequent addition of undiluted human serum showed a well defined conductance increase in NWl. These results clearly demon strate the detection of cancer markers with high sensitivity and selectivity in human serum. Telomerase detection and activity ● dNTPs To define further the potential of our silicon-nanowire arrays as cancer diagnostic tools, we investigated a nucleic acid-based marker assay involving detection of telomerase. Telomerase is a eukaryotic ribonu- cleoprotein complex,4 that catalyzes the addition of the telomeric repeat sequence TTAGGG to the ends of chromosomes using its intrinsic RNA as a template for reverse transcription334.Telomerase known human cancers >,o, but no activity was found in most of the adjacent somatic tissues, and telomerase has thus been proposed as a marker for cancer g Conductance-versus-time measurements recorded simultaneously detection and as a therapeutic target. e from NWl and NW2 as different donkey serum solutions were The telomerase assay is illustrated in Figure 4. First, silicon o sequentially delivered to the devices are shown in Figure 3c,d Delivery nanowire device elements within an array are functionalized with E of donkey serum containing 59 mg/ml total protein did not lead to an oligonucleotide primers complementary to the telomerase bindi appreciable conductance change relative to the standard assay buffer. site. Second, the presence or absence of telomerase is then detected by Donkey serum solutions containing f-PSA led to concentration- monitoring the nanowire conductance after delivery of a sample cell a dependent conductance increases only for NWl; no conductance extract to the device array. In the case of p-type nanowire device g changes were observed in NW2(Figs. 3c, d). Well-defined conductance elements, binding should reduce the conductance, as telomerase changes were observed for PSA concentrations as low as 0.9 pg/ml, (pl 10)7 is positively charged at physiological pH. Third,addition hich corresponds to a concentration 100-billion times lower than of deoxynucleotide triphosphates(dNTPs) leads, in the presence of hat of the background serum proteins. Similar results were obtained telomerase, to elongation, which should produce an increase in for human serum samples( Fig. 3e). Specifically, addition of undiluted conductance owing to the incorporation of negatively charge o human serum, which contains f-PSA, blocked with an excess of Abl, nucleotides near the nanowire surface a b 880 1.020 Time(s) Figure 5 Detection of telomerase (a)Conductance-versus-time data recorded for oligonucleotide-modified p-type silicon-nanowire devices, after the introduction of (l) a solution containing extract from 100 HeLa cells and 0. 4 mM dcTP, (2)a mixture all four dntPs(dATP, dgtP, duTP and dcTP)each at O 1 mM, (3)a solution containing extract from 100 HeLa cells and 0. 4 mM dCTP and (4)0.4 mM dCTP only Points()and (4)were recorded using a second device. ( b)Conductance-versus time data recorded for oligonucleotide-modified nanowire device after delivery of (1) a solution containing extract from 100,000 normal human fibroblast cells and 0. 4 mM dcTP, (2)a mixture of all four dntPs each at o 1 mM, (3)a solution containing extract from 10,000 HeLa cells, 0.4 mM dCTP, and 5 uM oligonucleotide(sequence: 5-TTTTITAATCCGTCGAGCAGAGTT-3), (4)a mixture of all four dNTPs each at 0.1 mM, (5)a solution containing extract from 10, 000 heat-deactivated HeLa cells(90C, 10 min) and 0.4 mM dCTP and(6)a mixture of all four dNTPs at 0. 1 mM. (c)Conductance-versustime data recorded on a p-type silicon- nanowire device after the introduction of (1)a solution containing extract 100 HeLa cells and 0. 4 mM dCTP, and (2)a mixture of all four dNTPs each at o1 mM and 20 uM AZTTP. Inset: Plot of the inhibition of elongation AZTTP concentration, where 100% corresponds to conductance change associated with elongation in the absence of AZTTP VOLUME 23 NUMBER 10 OCTOBER 2005 NATURE BIOTECHNOLOGYConductance-versus-time measurements recorded simultaneously from NW1 and NW2 as different donkey serum solutions were sequentially delivered to the devices are shown in Figure 3c,d. Delivery of donkey serum containing 59 mg/ml total protein did not lead to an appreciable conductance change relative to the standard assay buffer. Donkey serum solutions containing f-PSA led to concentration￾dependent conductance increases only for NW1; no conductance changes were observed in NW2 (Figs. 3c,d). Well-defined conductance changes were observed for PSA concentrations as low as 0.9 pg/ml, which corresponds to a concentration B100-billion times lower than that of the background serum proteins. Similar results were obtained for human serum samples (Fig. 3e). Specifically, addition of undiluted human serum, which contains f-PSA, blocked with an excess of Ab1, showed little change in baseline for either NW1 or NW2, although subsequent addition of undiluted human serum showed a well￾defined conductance increase in NW1. These results clearly demon￾strate the detection of cancer markers with high sensitivity and selectivity in human serum. Telomerase detection and activity To define further the potential of our silicon-nanowire arrays as cancer diagnostic tools, we investigated a nucleic acid–based marker assay involving detection of telomerase. Telomerase is a eukaryotic ribonu￾cleoprotein complex31,32 that catalyzes the addition of the telomeric repeat sequence TTAGGG to the ends of chromosomes using its intrinsic RNA as a template for reverse transcription33,34. Telomerase activity has been found in at least 80% of all known human cancers35,36, but no activity was found in most of the adjacent somatic tissues, and telomerase has thus been proposed as a marker for cancer detection and as a therapeutic target. The telomerase assay is illustrated in Figure 4. First, silicon￾nanowire device elements within an array are functionalized with oligonucleotide primers complementary to the telomerase binding site. Second, the presence or absence of telomerase is then detected by monitoring the nanowire conductance after delivery of a sample cell extract to the device array. In the case of p-type nanowire device elements, binding should reduce the conductance, as telomerase (pI B 10)37 is positively charged at physiological pH. Third, addition of deoxynucleotide triphosphates (dNTPs) leads, in the presence of telomerase, to elongation, which should produce an increase in conductance owing to the incorporation of negatively charged nucleotides near the nanowire surface. Telomerase dNTPs Figure 4 Schematic of the telomerase binding and activity assay. (Top) Silicon-nanowire devices modified with oligonucleotide primer. (Middle) A solution containing telomerase is delivered to the device array, and telomerase binds in a concentration-dependent process. (Bottom) Subsequent addition of dNTPs leads to telomerase-catalyzed primer extension/elongation. 960 950 1,060 100 80 60 40 % Elongation 20 0 0 5 10 [AZTTP] (µM) 15 20 1,040 1,020 1,000 980 940 930 920 910 900 0 200 400 600 800 1,000 0 200 400 600 800 1,000 1,200 880 1 1 1 3 4 5 6 2 2 2 3 4 800 720 0 400 Conductance (nS) 800 Time (s) Time (s) Time (s) 1,200 1,600 ab c Figure 5 Detection of telomerase (a) Conductance-versus-time data recorded for oligonucleotide-modified p-type silicon-nanowire devices, after the introduction of (1) a solution containing extract from 100 HeLa cells and 0.4 mM dCTP, (2) a mixture all four dNTPs (dATP, dGTP, dUTP and dCTP) each at 0.1 mM, (3) a solution containing extract from 100 HeLa cells and 0.4 mM dCTP and (4) 0.4 mM dCTP only. Points (3) and (4) were recorded using a second device. (b) Conductance-versus-time data recorded for oligonucleotide-modified nanowire device after delivery of (1) a solution containing extract from 100,000 normal human fibroblast cells and 0.4 mM dCTP, (2) a mixture of all four dNTPs each at 0.1 mM, (3) a solution containing extract from 10,000 HeLa cells, 0.4 mM dCTP, and 5 mM oligonucleotide (sequence: 5¢-TTTTTTAATCCGTCGAGCAGAGTT-3¢), (4) a mixture of all four dNTPs each at 0.1 mM, (5) a solution containing extract from 10,000 heat-deactivated HeLa cells (90 1C, 10 min) and 0.4 mM dCTP and (6) a mixture of all four dNTPs each at 0.1 mM. (c) Conductance-versus-time data recorded on a p-type silicon-nanowire device after the introduction of (1) a solution containing extract from 100 HeLa cells and 0.4 mM dCTP, and (2) a mixture of all four dNTPs each at 0.1 mM and 20 mM AZTTP. Inset: Plot of the inhibition of elongation versus AZTTP concentration, where 100% corresponds to conductance change associated with elongation in the absence of AZTTP. 1298 VOLUME 23 NUMBER 10 OCTOBER 2005 NATURE BIOTECHNOLOGY ARTICLES © 2005 Nature Publishing Group http://www.nature.com/naturebiotechnology